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Patrice Dion 

Chandra Shekhar Nautiyal 

Editors 



SOIL BIOLOGY 

Microbiology 
of Extreme Soil 



Soil Biology 

Volume 13 



Series Editor 

Ajit Varma, Amity Institute of Microbial Sciences, 

Noida, UP, India " 



Volumes published in the series 



Applied Bioremediation and Phytoremediation (Vol. 1) 
A. Singh, O.R Ward (Eds.) 

Biodegradation and Bioremediation (Vol. 2) 
A. Singh, O.R Ward (Eds.) 

Microorganisms in Soils: Roles in Genesis and Functions (Vol. 3) 
F. Buscot, A. Varma (Eds.) 

In Vitro Culture of Mycorrhizas (Vol. 4) 
S. Declerck, D.-G. Strullu, J.A. Fortin (Eds.) 

Manual for Soil Analysis - Monitoring and Assessing Soil 

Bioremediation (Vol. 5) 

R. Margesin, F. Schinner (Eds.) 

Intestinal Microorganisms of Termites and Other Invertebrates (Vol. 6) 
H. Konig, A. Varma (Eds.) 

Microbial Activity in the Rhizosphere (Vol. 7) 
K.G. Mukerji, C. Manoharachary, J. Singh (Eds.) 

Nucleic Acids and Proteins in Soil (Vol. 8) 
P. Nannipieri, K. Smalla (Eds.) 

Microbial Root Endophytes (Vol. 9) 

B.J.E. Schulz, C.J.C. Boyle, T.N. Sieber (Eds.) 

Nutrient Cycling in Terrestrial Ecosystems (Vol. 10) 
P. Marschner, Z. Rengel (Eds.) 

Advanced Techniques in Soil Microbiology (Vol. 11) 
A. Varma, R. Oelmiiller (Eds.) 

Microbial Siderophores (Vol. 12) 
A. Varma, S. Chicholkar (Eds.) 



Patrice Dion • Chandra Shekhar Nautiyal 
Editors 



Microbiology 
of Extreme Soils 



Foreword by John D. Rummel 



4y Spri 



ringer 



Professor Dr. Patrice Dion 

Departement de phytologie 

Pavilion Charles-Eugene Marchand 

1030, avenue de la Medecine 

Universite Laval 

Quebec (Quebec) G1V 0A6 

Canada 

E-mail: patrice.dion@plg.ulaval.ca 



Professor Dr. Chandra Shekhar Nautiyal 

National Botanical Research Institute 

Rana Pratap Marg 

Lucknow 226001 

India 

E-mail: csn@nbri.res. in 



ISBN 978-3-540-74230-2 



e-ISBN 978-3-540-74231-9 



Soil Biology ISSN: 1613-3382 

Library of Congress Control Number: 2007934829 

© 2008 Springer- Verlag Berlin Heidelberg 

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concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, 
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are liable to prosecution under the German Copyright Law. 

The use of general descriptive names, registered names, trademarks, etc. in this publication does not 
imply, even in the absence of a specific statement, that such names are exempt from the relevant 
protective laws and regulations and therefore free for general use. 

Cover design: WMXDesign GmbH, Heidelberg, Germany 

Printed on acid-free paper 5 4 3 2 10 



spnnger.com 



Foreword 



My auxiliaries are the dews and rains which water this dry soil, 
and what fertility is in the soil itself, which for the most part is lean and effete. 

- Henry David Thoreau, Walden Pond 

The concerns that Thoreau had about his beans were nothing to those that would 
face a similarly conscientious gardener in the Atacama Desert or on the planet 
Mars, where dews are rare, or frozen, and rains are extremely rare - or absent alto- 
gether. Yet we live in a time when an appreciation of the differences and similari- 
ties among soils (or regolith: no organics detected on Mars, as yet!) can provide a 
perspective on life at its most fundamental level: that of microbiology. 

Microbes are the Earth's finest chemists, and most prodigious chemical engineers. 
Beyond pure chemistry, they know tricks with electrons that would make any Silicon 
Valley chip designer blush with pride. And yet their size and association with human 
food (good) and diseases (bad) has for more than a century obscured their essential 
place in making the Earth a habitable planet for humans. One of the most interesting 
facets of this book is that we are shown those chemists at work in one of their most 
important habitats. Soils comprise both a pervasive environment on our planet and one 
of the most important (even most fruitful!) of habitats with respect to human survival. 
What the chapters of this book make clear is that extreme soils have lessons that reflect 
on our understanding of all natural soil processes, and that, as a site for scientific explo- 
ration, these soils have unique attributes that are worth of study in their own right. 

This volume provides an excellent introduction to the study of extreme soil micro- 
biology, and a variety of the challenging and fascinating environments that Earth-bound 
microbes face. Some are natural, and some are the result of human activity, and all 
of them have lessons to teach us about life's adaptations within the "extreme" hori- 
zons of terrestrial soils. What's more, each of these chapters (including the chapter 
on the soils of Mars by Ronald L. Crawford and David A. Newcombe) can give us 
insights into strategies that may make life possible beyond the safe confines of our 
present-day biosphere, to other worlds in this solar system and beyond. 

John D. Rummel Senior Scientist for Astrobiology 

NASA Headquarters 

Washington, DC 20546 

U.S.A 



Preface 



Two founding intuitions inspired this book. First comes the suggestion that "All 
soils are extreme, but some are more extreme than others". Indeed, as is discussed 
amply in the chapters that follow, heterogeneities and discontinuities in the phys- 
ico-chemical environment are a hallmark of soil systems, with the obligation for 
soil microbes to constantly reinvent themselves through metabolic control and 
adaptation. In addition to this tell-tale variable character, some soils, designated 
here as "extreme", harbor a constant, defining parameter that limits colonization by 
most organisms found elsewhere. The second inspiration for the present book is a 
sense of awe in view of the virtually limitless inventiveness of the microbial world. 
Investigating this diversity and how it developed has become a task fundamental to 
our understanding of the world. In no other province of human activity do more 
stories await to be told: each new day brings its microbiological marvels, and not 
a few of them come from the examination of soil micro-organisms. Hence, the 
present book proposes the concept of "extreme soil microbiology" to researchers and 
advanced students, as a means for organizing current knowledge and stimulating 
further developments. 

Technological advances and sociological changes are such that scientific mores 
keep evolving. If Charles Darwin was living today, who knows if he would not 
choose to publish his ideas as a one-page article in a top journal? Nevertheless, even 
today, there remain people to write and read scientific books. We believe that one 
reason for this is that broadening one's view, through the examination of a text of 
wide and extensive coverage, nurtures one's capacity for learning and reflection. 
Whereas scientific ideas seem to arise in the most unexpected manner, typically 
while jumping out of bed or at the movies, they are often elicited by a largely 
unconscious process involving scrambling and relating a vast corpus of notions. To 
serve in this process, those notions have to be timely and accurate. They also must 
be presented clearly, so as to trigger curiosity and stimulate the imagination. We 
hope that the reader will find the current book to fulfill these expectations. 

The book is organized into three sections. The first section, "Principles of 
Extreme Soil Microbiology" presents a conceptual framework for further compari- 
sons and coincident analyses of the various extreme soil systems. Chapter 1 provides 
an overview and places the other book chapters into perspective. Chapter 2 focuses 
on microbial communities from various types of extreme soils, presenting elements 



viii Preface 

that contribute to their structure and diversity. Chapter 3 considers evolutionary 
mechanisms taking place in extreme soils. That extreme soils ecosystems are 
exquisitely amenable to scientific exploration is eloquently advocated in Chapter 4. 
A recurrent theme in this first part of the book is the significance of extreme soils 
as representations and models for other terrestrial or extraterrestrial environments. 

In the second section, soils that owe their extreme character to natural processes 
are considered, with a description of the corresponding microbial communities, 
either observed or sometimes deduced or hypothesized. Chapter 5 shows that salinity 
is superimposed on other soil characters to shape selective pressure for colonizing 
organisms, which, in response, have developed traits that offer potential for bio- 
technology and bioremediation. Chapter 6 deals with organisms finding unlikely 
havens in areas where rain remains an abstract, literally evaporated notion. 
Permafrost soils are particularly fragile and, accordingly, generate preoccupations 
that are echoed in various sections of the book; their microbiology is the object of 
particular scrutiny in Chapter 7. Curious and unexpected phenomena are presented 
in Chapter 8, relating to the distribution of thermophilic Bacillus that colonize geo- 
thermal soils of the Antarctic. Peatlands, that provide a unique refuge to microbes 
driving peculiar biogeochemical cycles, are covered in Chapter 9. Chapters 10 and 
1 1 deal with life as it occurs or may occur well outside our current human range. 
Convincing evidence is provided for microbial functioning in an iron-rich subterra- 
nean environment, whereas some Martian environments protected from the deadly 
Sun may well meet life's requirements. 

The third section examines the results of human action on soil microbes, with an 
eye on future biological consequences as we continue remodelling our environ- 
ments. Many lessons are to be learned from how micro-organisms deal with 
hydrocarbon contamination in extremely cold Antarctic soils, as is discussed in 
Chapter 12. While offering quite a contrast, the hot Kuwaiti desert is also home to 
oil-degrading microbial communities, which Chapter 13 discusses. Fires, whether 
they be of short or long duration, present soil micro-organisms with particular chal- 
lenges, as is discussed in Chapter 14. Whereas hyperaccumulating plants have 
attracted much attention as colonizers of heavy metal-contaminated soils, Chapter 
15 stresses that such plants owe much of their properties to rhizospheric and endo- 
phytic micro-organisms. Finally, Chapter 16 examines fungal responses to radionu- 
clide contamination of soil, particularly in view of the Chernobyl accident. It should 
be remarked that the book subdivisions remain somewhat artificial, as some phe- 
nomena, particularly fires and presence of heavy metals, may be of natural or else 
anthropogenic origin. So the placement of chapters reflects the editors' prejudice 
and, perhaps more deeply, a human propensity to self-criticism. 

Except for residing on a small desert island with a single coconut tree and really 
nothing much to do, every human process entails some form of compromise. 
Preparing this book proved to be no exception, as a balance was sought between 
size and completeness. So it will perhaps be felt that some important topics are not 
covered, although every effort was made to provide a comprehensive coverage of 
the most prevalent extreme soil systems. For this and other failings of the book, the 
editors take the entire responsibility. 



Preface ix 

We have been privileged as book editors to count on the collaboration of 
dedicated and expert authors. We should like to thank them here for their profes- 
sionalism and enthusiasm, and also for their patience in dealing with our frequent 
and sometimes almost harassing demands. Our gratitude also goes to Professor Ajit 
Varma, for having believed in this project and for his continued support as the 
Series Editor, and to Dr. Jutta Lindenborn, Life Sciences Editorial at Springer, for 
her expert guidance throughout the book preparation process. We also wish to 
thank Cecile Gauthier, for helpful suggestions and help in the editing process. As 
this project reaches to a close, we are persuaded that the results will prove well 
worth the effort, and that the reader will share our amazement as, page after page, 
life thriving in seemingly inhospitable soils reveals its secrets. 

Patrice Dion Quebec City 

Chandra Shekhar Nautiyal Lucknow 

July, 2007 



Contents 



Part I Principles of Extreme Soil Microbiology 

1 The Microbiological Promises of Extreme Soils 3 

Patrice Dion 

2 Microbial Diversity, Life Strategies, and Adaptation 

to Life in Extreme Soils 15 

Vigdis Torsvik and Lise 0vreas 

3 Extreme Views on Prokaryote Evolution 45 

Patrice Dion 

4 Biodiversity: Extracting Lessons from Extreme Soils 71 

Diana H. Wall 

Part II Natural Extreme Soils 

5 Halophilic and Halotolerant Micro-Organisms from Soils 87 

Antonio Ventosa, Encarnacion Mellado, Cristina Sanchez-Porro, 

and M. Carmen Marquez 

6 Atacama Desert Soil Microbiology 117 

Benito Gomez-Silva, Fred A. Rainey, Kimberley A. Warren-Rhodes, 
Christopher P. McKay, and Rafael Navarro-Gonzalez 

7 Microbial Communities and Processes in Arctic Permafrost 

Environments 133 

Dirk Wagner 

8 Aerobic, Endospore-Forming Bacteria from Antarctic 

Geothermal Soils 155 

Niall A. Logan and Raymond N. Allan 



xii Contents 

9 Peatland Microbiology 177 

Shwet Kamal and Ajit Varma 

10 Subsurface Geomicrobiology of the Iberian Pyritic Belt 205 

Ricardo Amils, David Fernandez-Remolar, Felipe Gomez, 

Elena Gonzalez-Toril, Nuria Rodriguez, Carlos Briones, 
Olga Prieto-Ballesteros, Jose Luis Sanz, Emiliano Diaz, 
Todd O. Stevens, Carol R. Stoker, the MARTE Team 

11 The Potential for Extant Life in the Soils of Mars 225 

Ronald L. Crawford and David A. Newcombe 

Part III Anthropogenic Extreme Soils 

12 Bacteriology of Extremely Cold Soils Exposed 

to Hydrocarbon Pollution 247 

Lucas A.M. Ruberto, Susana C. Vazquez, and Walter P. Mac Cormack 

13 Microbiology of Oil-Contaminated Desert Soils and Coastal 

Areas in the Arabian Gulf Region 275 

Samir Radwan 

14 Microbial Communities in Fire-Affected Soils 299 

Christopher Janzen and Tammy Tobin-Janzen 

15 Endophytes and Rhizosphere Bacteria of Plants Growing 

in Heavy Metal-Containing Soils 317 

Angela Sessitsch and Markus Puschenreiter 

16 Interactions of Fungi and Radionuclides in Soil 333 

John Dighton, Tatyana Tugay, and Nelli Zhdanova 

Index 357 



Contributors 



Aguilera, Angeles 

Centro de Astrobiologia (INTA-CSIC), Torrejon de Ardoz, Spain 

Allan, Raymond N. 

Department of Biological and Biomedical Sciences, Glasgow Caledonian 

University, Cowcaddens Road, Glasgow G4 OB A, U.K. R.Allan@gcal.ac.uk 

Amils, Ricardo 

Centro de Astrobiologia (INTA-CSIC), 28850 Torrejon de Ardoz, Madrid, Spain, 
Centro de Biologia Molecular (UAM-CSIC), Cantoblanco, 28049 Madrid, Spain, 
ramils @ cbm.uam.es 

Briones, Carlos 

Centro de Astrobiologia (INTA-CSIC), 28850 Torrejon de Ardoz, Madrid, Spain, 

brioneslc@inta.es 

Cannon, Howard 

NASA Ames Research Center, Mountain View, CA, USA 

Crawford, Ronald L. 

Environmental Biotechnology Institute, University of Idaho, Moscow, ID 83844, 

USA, crawford@uidaho.edu 

Davila, Fidel 

Centro de Astrobiologia (INTA-CSIC), Torrejon de Ardoz, Spain 

Diaz, Emiliano 

Centro de Biologia Molecular (UAM-CSIC), Cantoblanco, 28049 Madrid, Spain, 

eediaz @ cbm.uam.es 

Dighton, John 

Rutgers University Pinelands Field Station, New Lisbon, NJ 08064, USA, 

dighton @ camden.rutgers.edu 

Dion, Patrice 

Departement de phytologie, Pavilion Charles-Eugene-Marchand, 1030, 

avenue de la Medecine, Universite Laval, Quebec (Quebec) G1V 0A6, Canada, 

patrice.dion@plg.ulaval.ca 



xiv Contributors 

Dunagan, Steven 

NASA Ames Research Center, Mountain View, CA, USA 

Fairen, Alberto G. 

Centro de Biologia Molecular (UAM-CSIC), U. Autonoma de Madrid, Madrid, 

Spain 

Fernandez-Remolar, David 

Centro de Astrobiologia (INTA-CSIC), 28850 Torrejon de Ardoz, Madrid, Spain, 

fernandezrd@inta.es 

Glass, Brian 

NASA Ames Research Center, Mountain View, CA, USA 

Gomez, Felipe 

Centro de Astrobiologia (INTA-CSIC), 28850 Torrejon de Ardoz, Madrid, Spain, 

gomezgf@inta.es 

Gomez-Elvira, Javier 

Centro de Astrobiologia (INTA-CSIC), Torrejon de Ardoz, Spain 

Gomez-Silva, Benito 

Instituto del Desierto y Unidad de Bioquimica, Facultad Ciencias de la Salud, 

Universidad de Antofagasta, Casilla 170, Antofagasta, Chile, bgomez@uantof.cl 

Gonzalez-Toril, Elena 

Centro de Astrobiologia (INTA-CSIC), 28850 Torrejon de Ardoz, Madrid, Spain, 

etoril@cbm.uam.es 

Janzen, Christopher 

Chemistry Department, Susquehanna University, Selinsgrove, PA 17870, USA, 

janzen@ susqu.edu 

Kamal, Shwet 

Amity Institute of Microbial Sciences, Amity University Uttar Pradesh, 

Noida 201303, India, skamal@amity.edu 

Lemke, Lawrence G. 

NASA Ames Research Center, Mountain View, CA, USA 

Logan, Niall A. 

Department of Biological and Biomedical Sciences, Glasgow Caledonian 

University, Cowcaddens Road, Glasgow G4 0B A, UK, nalo@gcal.ac.uk 

Lynch, Kennda 

NASA Johnson Space Center, Houston, TX, USA 

Mac Cormack, Walter P. 

Instituto Antartico Argentino, Departamento de Biologia. Cerrito 1248, 

C1010AAZ, Buenos Aires, Argentina, wmac@huemul.ffyb.uba.ar 



Contributors xv 

Marquez, M. Carmen 

Department of Microbiology and Parasitology, Faculty of Pharmacy, University 

of Sevilla, 41012 Sevilla, Spain, cmarquez@us.es 

McKay, Christopher P. 

Space Science Division, NASA Ames Research Center, Moffett Field, CA 

94035-1000, USA, cmckay@mail.arc.nasa.gov 

Mellado, Encarnacion 

Department of Microbiology and Parasitology, Faculty of Pharmacy, University 

of Sevilla, 41012 Sevilla, Spain, emellado@us.es 

Navarro-Gonzalez, Rafael 

Laboratorio de Quimica de Plasmas y Estudios Planetarios, 

Instituto de Ciencias Nucleares, Universidad Nacional 

Autonoma de Mexico, Apartado Postal 70-543, Mexico D.F. 04510, 

Mexico, navarro@nucleares.unam.mx 

Newcombe, David 

Environmental Science Program, University of Idaho, 721 Lochsa Street, Suite 3, 

Post Falls, ID 83854, USA, dnewcombe@uidaho.edu 

0vreas, Lise 

Department of Biology, University of Bergen, P. Box 7800, Jahnebakken 5, 

N-5020 Bergen, Norway, lise.ovreas@bio.uib.no 

Parro, Victor 

Centro de Astrobiologia (INTA-CSIC), Torrejon de Ardoz, Spain 

Prieto-Ballesteros, Olga 

Centro de Astrobiologia (INTA-CSIC), 28850 Torrejon de Ardoz, Madrid, Spain, 

prietobo@inta.es 

Puschenreiter, Markus 

Department of Forest and Soil Sciences, University of Applied 
Life Sciences and Natural Resources, A-1190 Vienna, Austria, 
markus .puschenreiter @ boku . ac . at 

Radwan, Samir 

Department of Biological Sciences, Faculty of Science, Kuwait University, 

P.O. Box 5969, Safat 13060, Kuwait, radwan@kuc01.kuniv.edu.kw 

Rainey, Fred A 

Department of Biological Sciences, 202 Life Sciences Building, Louisiana State 

University, Baton Rouge, LA 70803, USA, frainey@lsu.edu 

Rodriguez, Nuria 

Centro de Astrobiologia (INTA-CSIC), 28850 Torrejon de Ardoz, Madrid, Spain, 

nrodriguez @ cbm.uam.es 



xvi Contributors 

Ruberto, Lucas A.M. 

Catedra de Microbiologic Industrial y Biotecnologia, Facultad de Farmacia y 
Bioquimica, Universidad de Buenos Aires. Junin 956, C1113AAD, Buenos Aires, 
Argentina, lruberto@ffyb.uba.ar 

Sanchez-Porro, Cristina 

Department of Microbiology and Parasitology, Faculty of Pharmacy, University 

of Sevilla, 41012 Sevilla, Spain, sanpor@us.es 

Sanz, Jose Luis 

Centro de Biologia Molecular (UAM-CSIC), Cantoblanco, 28049 Madrid, Spain, 

joseluis.sanz@uam.es 

Sessitsch, Angela 

Austrian Research Centers GmbH, Department of Bioresources, A-2444 

Seibersdorf, Austria, angela.sessitsch@arcs.ac.at 

Souza-Egipsy, Virginia 

Centro de Astrobiologia (INTA-CSIC), Torrejon de Ardoz, Spain 

Stevens, Todd O. 

Department of Biology, Portland State University, Portland, OR, USA, 

tstevens@gorge.net 

Stoker, Carol R. 

NASA Ames Research Center, Mountain View, CA, USA, cstoker@mail.arc.nasa.gov 

Tobin-Janzen, Tammy 

Biology Department, Susquehanna University, Selinsgrove, PA 17870, USA, 

tobinj an @ susqu.edu 

Torsvik, Vigdis 

Department of Biology, University of Bergen, P. Box 7800, Jahnebakken 5, 

N-5020 Bergen, Norway, vigdis.torsvik@bio.uib.no 

Tugay, Tatyana 

Department of Physiology and Taxonomy of Micromycetes, Institute 
of Microbiology and Virology, Ukrainian National Academy of Sciences, 
154 Zabolotny Street, Kiev 252143, Ukraine, tatyanatugay@rambler.ru 

Varma, Ajit 

Amity Institute of Microbial Sciences, Amity University Uttar Pradesh, 

Noida 201303, India, ajitvarma@aihmr.amity.edu 

Vazquez, Susana C. 

Concejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), 

Buenos Aires, Argentina, svazquez@ffyb.uba.ar 

Ventosa, Antonio 

Department of Microbiology and Parasitology, Faculty of Pharmacy, University 

of Sevilla, 41012 Sevilla, Spain, ventosa@us.es 



Contributors xvii 

Wagner, Dirk 

Alfred Wegener Institute for Polar and Marine Research, Research Unit Potsdam, 

Telegrafenberg A45, 14473 Potsdam, Germany, Dirk.Wagner@awi.de 

Wall, Diana H. 

Department of Biology and Natural Resource Ecology Laboratory, Colorado State 

University, Fort Collins, CO 80523-1499, USA, Diana@nrel.colostate.edu 

Warren-Rhodes, Kimberley A. 

Space Science Division, NASA Ames Research Center, Moffett Field, CA 

94035-1000, USA, kwarren-rhodes@mail.arc.nasa.gov 

Zavaleta, Jhony 

NASA Ames Research Center, Mountain View, CA, USA 

Zhdanova, Nelli 

Department of Physiology and Taxonomy of Micromycetes, Institute of 

Microbiology and Virology, Ukrainian National Academy of Sciences, 

154 Zabolotnoy Street, Kiev 252143, Ukraine, zhdanova_imv_ua@rambler.ru 



Chapter 1 

The Microbiological Promises of Extreme Soils 



Patrice Dion 



1.1 Introduction 

Whereas the notion of extreme environment has received much attention from 
microbiologists, this generalization does not systematically include extreme 
soils. There may be at least two reasons for this. First, any soil may be considered 
as extreme for the colonizing microbes constantly facing starvation, desiccation, 
predation, and other attacks. In this sense, the notion of "extreme soil" would 
appear pleonastic. A second reason for the uncommon use of the term "extreme 
soil" might be the opinion that there is little to be gained from it, inasmuch as 
every soil has its particularities and, in its very nature, is refractory to human 
efforts at unification and simplification. In this sense, any grouping of soils from, 
say, the Antarctic or hot deserts into a common category designated as "extreme" 
would appear futile, if not detrimental to a precise understanding of soils and 
their microbial populations. However, one might take the stance that, although it 
certainly serves to be aware of these difficulties, there is still much to be learned 
from running into them. Hence, it is hoped that the present book will be a dem- 
onstration of the usefulness of the extreme soil concept to microbiologists. 

Various extreme soils have been the topic of numerous and fruitful studies deal- 
ing with the characterization of microbial communities and processes. These stud- 
ies have done much to enrich our understanding of microbial diversity and of 
biogeochemical and other biological mechanisms. They allow us to grasp microbial 
adaptability and to envision practical applications. Reading the enclosed collection 
of chapters will make it clear that unifying these studies under the general theme of 
"extreme soils," and associating this concept with the broader notion of "extreme 
environments" leads to essential theoretical and practical advances. 

We qualify a soil as "extreme" when it supports colonization by organisms pre- 
senting a specific and common adaptation. The specifying character of an extreme 
soil may be physical in nature, and correspond to extreme values of temperature, or 



Patrice Dion 

Departement de phytologie, Pavilion Charles-Eugene Marchand, 1030, avenue de la Medecine, 

Universite Laval, Quebec (Quebec), Canada G1V 0A6 

e-mail: patrice.dion@plg.ulaval.ca 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 
© Springer- Verlag Berlin Heidelberg 2008 



4 P. Dion 

to exposure to radiation or intense heat. Alternatively, it may also be defined in 
chemical terms, with the salt content or the presence of toxic pollutants exerting a 
preponderant influence on microbial processes. The extreme character may be con- 
ferred on soils by climatic, geological, or other environmental factors, or else by 
human activities. One might observe here that the distinction between nature-driven 
and anthropogenic processes is becoming increasingly blurred, as a result of our 
improved capacity to relate effects to their cause, and also as human activities exert 
an ever stronger influence on an expanding scale. 

The chapters included in this book provide a careful description of microbial 
communities exposed to various soil-borne challenges. Bringing together analyses 
of a wide range of soil systems invites comparative assessments and well-founded 
extrapolations. Thus, it is hoped that, in addition to providing timely knowledge 
about extreme soil microbial dwellers, the book will pay tribute to a vast and largely 
unexplored territory wide open to microbiological enquiry. Indeed, extreme soils 
promise crucial progress in our understanding of microbial activities and adaptation 
processes, as well as in our ability to rationally influence ecosystems upon which 
terrestrial life depends. 



1.2 Extreme Soils and Microbial Community Structure 
and Evolution 

Following an unprecedented search for marine microbial sequences, an immense 
diversity of marine bacteria was revealed, with at least 25,000 different types of 
micro-organisms being estimated to exist per litre of seawater. The methods used 
in the study made it possible to relate intraribotype genetic variation to environmen- 
tal factors. Species may be organized into subtypes, and the corresponding variation 
results from physical barriers, short-term stochastic effects, and functional differen- 
tiation acting in combination (Rusch et al. 2007). Comparison of marine and ter- 
restrial organisms showed that 68% of the nearly 7,000 examined protein domains 
varied between the two classes of micro-organisms, this variation being in part the 
result of different metabolic requirements for marine and terrestrial life (Yooseph 
et al. 2007). These results are a testimony to adaptability of microbial life. 

Extreme soils offer us an opportunity to understand microbial diversity as an 
adaptive response that both reflects and multiplies environmental diversity. Indeed, 
the soil can be hot or cold, acidic or basic, wet or dry, saline, radioactive, polluted 
with heavy metals or hydrocarbons, or located on Earth or some other celestial 
body. Soil microbes pay tribute to this multiplicity of characters by undergoing and 
maintaining diversification (see Chapter 2). 

Extreme environments present peculiar evolutionary challenges to prokaryotes, to 
which might correspond peculiar evolutionary responses. Identification of these spe- 
cificities may reveal hitherto unnoticed aspects of evolutionary processes. In particu- 
lar, extreme conditions might force evolution of traits that are otherwise invariant. 
More generally, studies on extremophile evolution may help shed light on the roles of 



1 The Microbiological Promises of Extreme Soils 5 

environmental pressure in driving evolution. Indeed, competition between individuals 
may play a larger role in nonextreme environments, whereas environmental pressures 
would be determinant under extreme conditions. Soils are physically constituted by 
the orderly collection of sizable and interacting structures. Physicochemical parame- 
ters are superimposed on this primary framework. Microbial niches and correspond- 
ing diversity are defined by a series of combinatorial operations that the various 
organizational levels of the soils render possible (see Chapter 3). 

In comparing microbial communities of nonextreme and extreme soils, it becomes 
apparent that extreme soil communities reach unique equilibria, corresponding to 
under- or overrepresentation of certain community components. The degree to which 
community member exclusions and inclusions occur, and the nature of these proc- 
esses, vary in different extreme soils. Attempting a synthesis of these particular 
responses may lead to identification of crucial microbial mechanisms for survival, 
growth, dissemination, and adaptation. Such a synthesis may also provide insights on 
the forces at play to shape and structure biological communities in extreme as well as 
nonextreme soils (see Chapter 4). Biogeochemical cycles, as they operate under 
extreme conditions, bring into sharp focus important aspects of microbial community 
functioning and bear direct relevance to global equilibria (see Chapters 7, 9, and 14). 



1.3 Extreme Soils and Microbial Physiology 

Comparisons of microbial adaptations in extreme soils and other extreme environ- 
ments suggest commonalities in physiological processes and cell adaptations. For 
example, patterns of adaptation to heat and cold, through adjustments in protein 
thermal stability and membrane composition (see Chapters 2, 3, and 8), are similar 
in extreme soils as in other environments. Also, compatible solutes contribute to 
maintain osmotic balance in halophilic organisms from saline soils and other saline 
environments (see Chapter 5). Such a pattern of common adaptations, superimposed 
on additional and specific adaptations to soil, water, or other environments, is sugges- 
tive of modularity in microbial evolutionary processes. Modules can be defined as 
"building blocks of interacting elements that operate in an integrated and relatively 
autonomous manner" (Schlosser 2004). Modules may be thought of as structures, 
but also as processes, that would be articulated according to three principles. These 
principles are: (1) connectivity, meaning that they are triggered in a switchlike fash- 
ion by a variety of inputs; (2) hierarchization, implying that modules may be spatio- 
temporally embedded in higher-order modules, or overlap by sharing common 
elements; and (3) multiple instantiation, which contributes to delimitate a module 
from others of the same type by its independent perturbability during development. 
Specifically, modules of evolution are units of integrated and context-insensitive 
evolutionary changes (Schlosser 2004). From these considerations, it appears that 
adaptive processes in extreme soil bacteria may be modular, in the sense that adapta- 
tions to the soil and extreme components of the environment may occur somewhat 
independently, while influencing each other through epistasis (see Chapter 3). 



6 P. Dion 

It might be worth mentioning in this respect that relatively little is known on physi- 
ology of life in arid environments and the adaptations involved. In most studies, water 
stress refers to external solute excess, rather than dehydration. Whereas salt and water 
stress often come together (Wierzchos et al. 2006), and may be both dealt with 
through osmotic adjustment (see Chapter 2), it is possible to distinguish between tol- 
erance to salt and to water in plants (Munns 2002), and also perhaps in micro-organisms 
(Zahran 1999). Most studies where dehydration is considered refer to food (Grant 2004). 
Studies on arid soils deal mainly with community structure (McKay et al. 2003; see 
also Chapter 6) and little is known about microbial physiology and adaptations, as 
many of the dominant organisms in arid soils yet remain to be isolated (Drees et al. 
2006). One aspect that is being studied is the physiology of photosynthesis in cyano- 
bacteria colonizing arid soils (Luttge et al. 1995; Ohad et al. 2005). It is also known 
that, in Deinococcus radiodurans, radiation and desiccation resistance are correlated 
(Mattimore and Battista 1996), and related at least in part to a remarkable capacity 
for reassembly of broken chromosomes (Zahradka et al. 2006). 



1.4 Microbial Functions in Extreme Soils 

Modularity, which occurs at the level of cell adaptation to the extreme and soil 
environmental components, may also be identified as a defining factor of soil microbial 
communities, where it arises as a consequence of redundancy and complementarity 
of soil functions (Nannipieri et al. 2003). Redundancy allows microbial groups to 
be considered as equivalent and interchangeable with respect to function, thus buff- 
ering biogeochemical cycling and other ecosystem processes against restricting 
changes, such as selection and metabolic tradeoffs. 

The extreme character acts on redundancy, diminishing it without altering ecosystem 
processes (see Chapter 4). This results in a strengthening of the correspondence between 
identity and function, and leads to a better illustration of microbial function as it more 
directly relates to particular microbial types. Clues to this simplifying, looking-glass 
effect are proposed in this volume, and are provided, for example, by observations on 
dissimilatory sulfate reduction by certain halophiles in saline soils and their role in eco- 
system functioning (see Chapter 5), hydrocarbon degradation as it is performed by per- 
mafrost microbial communities (see Chapter 12), and involvement of endo- and 
ecto-mycorrhizae in radionuclide uptake and transfer to plants (see Chapter 16). It may 
well be that further studies on these and other systems will highlight the potential of 
extreme soils as simplified objects for study of microbial processes. 



1.5 Practical Value of Extreme Soils 

Like a cat, the Earth cleans itself continuously, although there is growing concern 
that autogenous processes may not suffice given the extent of human-inflicted 
damage. Soil contamination occurs worldwide, and it is striking that bacteria 



1 The Microbiological Promises of Extreme Soils 7 

from pristine Arctic soils express mer (or mercury resistance) genes (Poulain 
et al. 2007). 

One might consider the degradation or elimination of pollutants as yet another 
manifestation of overall soil productivity, and then be led to suggest that the intrin- 
sic cleanup potential will be less in extreme soils, as compared to nonextreme soils. 
This suggestion arises from the conjunction of two observations, which are, first, 
that microbial diversity is often lower in extreme soils than in nonextreme soils (see 
Chapter 2), and, second, that ecosystem productivity increases with biodiversity 
(Tilman 1999). On the other hand, extreme soils might be more responsive than 
nonextreme soils to application of exogenous microbial inoculant with remediating 
activity, as lower diversity might make communities more susceptible to invasions 
(Tilman 1999). 

The relationship between diversity and stability of an ecosystem is the object of 
some controversy and may be influenced by species composition (Bezemer and van 
der Putten 2007; Tilman et al. 2007). This may explain why attempts at accelerating 
the degradation of oil pollutants in Antarctic soils through bioaugmentation are 
often unsuccessful (see Chapter 12). 

Conceptually, two situations may arise, with the extreme soil parameter being 
the direct object of the bioremediation process or simply acting as an intrinsic con- 
founding factor. Attempts to use bacteria to reduce the impact of salts (Bacilio et al. 
2004; Ashraf et al. 2006) or heavy metals (see Chapter 15), belong to the first cate- 
gory, and, in this case, the extreme character of the soil will become attenuated 
following successful treatment. Bioremediation efforts of hydrocarbon-contaminated 
cold (see Chapter 12) or arid (see Chapter 13) soils fall into the second class of 
bioremediation interventions. 

Extremophiles offer an ever- expanding domain to biotechnological applications 
(Podar and Reysenbach 2006), and extreme soils represent a rich and still relatively 
unexplored source of useful microbes. For example, halophiles colonizing saline 
soils produce a wealth of macromolecules and small compounds of potential use 
(see Chapter 5). 



1.6 Extreme Soils and the Boundaries of Life 

Although there are sound and totally unemotional reasons to send humans into 
space instead of strictly relying on robots (Crawford 2004), the fascination exerted 
by human expansion through the Earth and beyond points to additional motivations. 
In yielding to our relentless drive to expand, we are doing little more than applying 
virtually unlimited imagination and technical skills to satisfy our innate desire at 
self-perpetuation. Along the process, and especially since the end of the Cold War, 
we are conducted to establish the pacifying image of humanity as a constant creator 
of knowledge and settings. Not surprisingly then, life as it is envisioned elsewhere 
in the cosmos is often portrayed as akin to Earthly biological processes (Pace 
2001), which is certainly a reasonable view as long as we restrict our exploratory 
range to our immediate planetary neighbors. Looking farther beyond, a variety of 



8 P. Dion 

other chemistries based on liquids other than water may be envisioned (Bains 2004; 
Benner et al. 2004). 

Following its formation 4.6 billion years ago and early bombardment, Mars is 
believed to have been through three geologic ages, the first of which, termed the 
phyllosian era, coincided with a nonacidic aqueous alteration of planetary material. 
The planet might have been habitable during this period, which ended 3.5 billion 
years ago, when surface water became increasingly acidic during the theiikian era, 
and then disappeared to initiate the siderikian era, that is still going on today 
(Bibring et al. 2006). However, liquid water might have resided very close to the 
surface throughout Mars' history, as is indicated by the observation of gullies filled 
during the last decade with a liquid that might be water (Malin et al. 2006). These 
observations leave open the possibility that Martian life, if indeed it was initiated 
during the Earth-like phyllosian era, might have been maintained in isolated subter- 
ranean oases up to this day (see Chapter 11). 

The eventual demonstration and examination of extraterrestrial life will have an 
unimaginable impact on human thinking. It appears, however, that the mere search 
for this life is already bearing fruit. It leads us to systematically consider how life 
can be recognized and studied (see Chapter 1 1). It provides an incentive to investi- 
gate Mars-like ecosystems of our planet, such as sun-bathed deserts (see Chapter 6) 
or rocks where metabolic energy is extracted from metal chemistry (see Chapter 
10). It also leads us to reflect upon and better comprehend ecological processes 
upon which our lives immediately depend (Wilkinson 2003). Ultimately, we are 
presented with pictures of our past and our future (see Sections 1.7 and 1.8) and, 
consequently, our very nature. 

While reflecting on the nature of life, Erwin Schrodinger wrote: "Living matter, 
while not eluding the 'laws of physics' as established up to date, is likely to involve 
'other laws of physics' hitherto unknown, which, however, once they have been 
revealed, will form just as integral a part of this science as the former" (Schrodinger 
1944). Although this view has been amply commented and criticized (Sarkar 1991), 
it may retain some intuitive value, in the sense that we have entrenched the notion 
that our current physical knowledge does not fully account for life, even as we 
know it in its most current manifestations. The history of biological enquiry may 
be viewed as an endless tentative to restrict and even contradict this notion of life 
as escaping common physical laws. 

Today, we maintain a desire to define life from the outside, that is, to determine 
where life can and cannot exist and then identify absolute differences between these 
two classes of environments. Certainly, extremely arid (see Chapters 6 and 13) or 
cold (see Chapters 7 and 12) land may be viewed as a patchwork of life-prone and 
life-hostile zones, as does extraterrestrial soil (see Chapter 11). For its part, isola- 
tion might not impede life development itself, but rather narrow its manifestations 
(see Chapter 8). In gaining such a sub tractive vision of life, in the sense of deter- 
mining where life cannot be and what factors restrict its range, we demonstrate the 
limitations of biological processes and adaptations. Thus, a frontier is drawn, 
within which life is thought to remain. Within this frontier composed of water and 
carbon, every known life-related phenomenon exists, from the citric acid cycle to 



1 The Microbiological Promises of Extreme Soils 9 

consciousness. However, lingering questions remain: can this frontier be pushed 
just a bit further? Is there really nothing living beyond it? 



1.7 Extreme Soils and Our Past 

Inspired by Beijerinck, Bass Becking has said that "Everything is everywhere, but 
the environment selects," and this statement has been the object of numerous com- 
ments (de Wit and Bouvier 2006). Certainly, some observations reported in this 
book, for example, on the presence of thermophilic endospore formers in cold soils 
and even Arctic ice (see Chapter 8), or of Rhodococcus strains with a capacity for 
hydrocarbon degradation in pristine Antarctic soils (see Chapter 12) seem to cor- 
roborate the notion of a universally shared microbial substratum for environmental 
selection. However, upon further consideration of this notion, two questions arise, 
which concern the nature of, first, the environmental selection implied in the state- 
ment from Bass Becking, and, second, the mechanisms that have allowed "every- 
thing" to have become present "everywhere." 

With respect to the first question, bacterial dispersal is likely to set the table for 
what has been termed "postimmigration evolution" (Novak 2007), that implies that 
selection of invasive species is accompanied by adaptive genotypic modifications. 
Furthermore, it appears that these two recognized components of evolutionary proc- 
esses, mutation and selection, are functionally related and environmentally determined, 
albeit to various extents. The mutation rate is influenced by various environmental 
components in addition to classical mutagens, this influence being exerted in the 
absence of any directionality to mutation events (see Chapter 3). Bacteria take differ- 
ent avenues towards genetic changes, that include horizontal gene transfer in addition 
to mutation. The ability to acquire foreign genes may itself be influenced by environ- 
mental parameters. This may occur indirectly, through the creation of hotspots of high 
bacterial density where plasmid transfer occurs at increased frequency (S0rensen et 
al. 2005), or even directly, by SOS-mediated enhancement of expression of genes 
involved in DNA transfer (Beaber et al. 2004). 

Dispersal, that may occur over considerable distances, comes immediately to 
mind as a possible explanation for the apparent ubiquity of bacteria. However, the 
notion of bacterial ubiquity must be put in perspective. Some bacterial species, 
including various plant and animal pathogens, are distributed worldwide, whereas 
others are endemic. Within cosmopolitan species, some particular genotypes may 
in fact be endemic. The ability for dispersal would be associated with the presence 
of particular genes (Ramette and Tiedje 2007a). Reflecting this variation in distri- 
bution range, bacterial populations exhibit clear spatial patterns that arise both from 
environmental heterogeneity and from spatial distance taken as a measure of past 
historical events and disturbances. Within- species diversity may be influenced by 
spatial distance as well as by environmental heterogeneity, whereas species abun- 
dance and the composition of communities are more influenced by local environ- 
mental interactions. However, a considerable proportion of bacterial diversity 



10 P. Dion 

remains unexplained by the currently measured variables (Ramette and Tiedje 
2007b). Because they represent well-defined and localized environments, extreme 
soils may prove valuable objects of study in bacterial biogeography. For example, 
suitable and unsuitable habitats for extant soil communities in the Antarctic Dry 
Valleys are defined by a combination of factors that include current and historical 
elements. Among the latter are past climates, which influence the level of lakes and 
glaciers. Also of relevance are legacies of productivity from past ecosystems that 
influence the chemistry of contemporary soils and their organic matter (Virginia 
and Wall 1999; see also Chapter 4). 



1.8 Extreme Soils and Our Future 

Very few areas of our planet now escape human influence, and the global environ- 
ment may be described as the "human-made context of our lives" (Dalby 2002). 
Nearly half of the land surface has been transformed by direct human action (Steffen 
et al. 2004), and sometimes humans may cause catastrophic soil changes that extend 
over considerable time and space scales. The great Centralia coal fire and other simi- 
lar events are a case in point, and have considerable impact on soil microflora and 
other soil properties (see Chapter 14). Indirect human action, particularly through 
global climate change and what has been termed "global distillation" (or the spread 
of persistent organic pollutants) may be even more pervasive, with the result that vir- 
tually no pristine land is said to remain (Dalby 2002). Thus, we may be experiencing 
a global extremization of the soils (see Chapter 4), which may affect interactions 
between humans and a variety of soil-dependent or soil-residing organisms, would 
these particular organisms be sources of food or disease. 

Throughout its modern history, humankind has been able to escape the conse- 
quences of its actions by exporting, either its waste or its populations themselves, 
some less fortunate human groups occasionally suffering in the process as unwilling 
recipients. Now we have reached a point where these movements appear useless, with 
a radical transformation of air, water, and soil threatening the self-maintained balance 
upon which all life forms depend. Particularly worrisome is the spectre of a spiralling 
uncontrollable process, whereby environmental change becomes the trigger of ever 
greater and more damaging changes. Such self- sustained processes may eventually 
occur in peatlands, which have been acting as net carbon sinks and methane sources 
since the early Holocene. Desiccation of Siberian peatlands would elevate C0 2 con- 
centrations through peat oxidation but would reduce CH 4 emissions, with the out- 
come on net radiative balance being difficult to estimate. However, the balance may 
well tilt towards an intensification of greenhouse warming (Smith et al. 2004). This 
is one reason why microbial communities and processes of peatlands deserve special 
scrutiny (see Chapter 9). Permafrost soils, where carbon storage and methanogenesis 
also exist, are similarly preoccupying (see Chapter 7). 

Industrialized societies have been especially good at minimizing the impact of 
microbial pathogens, through a combination of sanitation and hygiene measures, 



1 The Microbiological Promises of Extreme Soils 1 1 

large-scale vaccination strategies, and heavy antibiotic use. Now there are indica- 
tions that this relatively disease-free era may be coming to an end. Global changes 
are expected to play a role in this resurgence of microbial pathogenicity, with the 
poorest nations being hit first and strongest. Soils are well-known reservoirs of 
microbial human pathogens (Santamana and Toranzos 2003), so that soil changes 
may well be associated with modifications in transmissibility and incidences of 
various diseases. 

Many of these changes as they relate to disease transmission concern dispersal. A 
striking example is provided by Vozrozhdeniye Island, which was once in the middle 
of the now drying Aral Sea, and hosted the main Soviet center for testing biological 
weapons, antidotes, and vaccines. Upon shutdown of the Soviet program in 1988, 
slurries of anthrax spores and other pathogens were buried on the island. Vozrozhdeniye 
has now grown from a 3 3 -km island to a 145 -km peninsula, and, with some of the 
anthrax spores having remained viable (Whish- Wilson 2002), has become the object 
of intense surveillance and decontamination programs. In addition to persisting 
anthrax, a military-grade, antibiotic-resistant strain of plague bacterium may also 
have survived among the rodent population in the testing range (Pala 2005). There is 
a possibility of short-term survival of Yersinia pestis in soils, embedded in flea feces 
or tissues of dead animals, and even of its persistence in soils in dormant form or as 
an intracellular parasite of soil protozoa (Gage and Kosoy 2005). 

The recent reconstruction of the 1918 Spanish flu epidemic virus, from the body 
of a flu victim buried in Alaskan permafrost (Tumpey et al. 2005), should also pro- 
vide us with an opportunity to ponder the infectious potential of currently inacces- 
sible or inert material. Although not documented still, the possibility exists that 
seemingly vanished pathogens, including the smallpox virus (Stone 2002), remain 
preserved in frozen state in permafrost burial sites. Hence, it appears that the study 
of permafrost-inhabiting microbial communities (see Chapters 7 and 12), while 
providing a remarkable insight into microbial adaptations and processes, may take 
unexpected relevance and offer twist endings. The possibility that some Archaea 
may be as of yet unrecognized human pathogens (Eckburg et al. 2003) also places 
soil microbial reservoirs and soil changes into a new perspective. 

Cioran tells us of Socrates, who, the day before he would die, was learning a 
tune on the flute. Being asked what good it would do to him, he answered "To know 
this tune before I die." Following the philosopher's example, we will continue 
learning about extreme soils and their microbial inhabitants. This will help us 
monitor the changes ahead. We will also be in a better position to cope with these 
changes, which it is hoped will not prove as dreadful as a cup of hemlock tea. 



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Chapter 2 

Microbial Diversity, Life Strategies, 

and Adaptation to Life in Extreme Soils 

Vigdis Torsvik(K) and Lise 0vreas 



2.1 Introduction: What Is an Extreme Environment? 

There is no general consensus on how to define an extreme environment. From an 
anthropocentric point of view, physicochemical conditions supporting mammalian 
life appear as normal, and conditions deviating from these are considered as 
extreme. However, what is extreme and what is normal for microbes remains debat- 
able, and the concept "extreme" as we use it may not necessarily be appropriate for 
micro-organisms (Gorbushina and Krumbein 1999). Micro-organisms dwell in vir- 
tually all types of soil habitats. These range from extremely dry and cold deserts in 
the Antarctic and deep into permafrost soils to geothermal and humid soils in vol- 
canic areas, from extremely acid mines with sulfuric acid to high alkaline areas. 
Microbial life can also exist in salt crystals, under extremely low water activity, and 
low nutrient concentrations. As a group, micro-organisms have the highest ability 
of all life forms to adapt to extreme and stressful environments. This includes new 
types of habitats created by anthropogenic activities, such as those polluted with 
heavy metals, radionuclides, and high concentrations of toxic xenobiotic com- 
pounds (e.g., polychlorinated biphenyls, hydrocarbons, and pesticides). 

Environments which we consider extreme can be inhabited by well-adapted 
microbiota, and if the environment is stable the resident micro-organisms may not 
experience any stress but metabolize and grow successfully under strictly limiting 
conditions, which appear normal to them. During the approximately 3.8 billion 
years that micro-organisms inhabited Earth, dramatic changes in physicochemical 
conditions of Earth's surface have periodically occurred. Thus, conditions that can 
be considered as "normal" for life have also changed. 

An alternative view is that any stable environment can be regarded as "normal," 
and that an extreme environment is one with highly fluctuating conditions where 
the organisms experience episodic or periodic dramatic environmental changes. In 
unstable and extreme environments, the metabolic costs to survive stress may be 



Vigdis Torsvik 

Department of Biology, Centre for Geobiology, University of Bergen, RBox 7800, Jahnebakken 5, N- 

5020 Bergen, Norway 

e-mail: vigdis.torsvik@bio.uib.no 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 15 

© Springer- Verlag Berlin Heidelberg 2008 



16 V. Torsvik and L. 0vreas 

high, and most organisms will probably die. However, some micro-organisms have 
very high physiological and ecological plasticity, which makes them well adapted 
to survive even in environments where the conditions may change suddenly and 
dramatically. Organisms that can tolerate considerable environmental stress caused 
by fluctuating conditions have been termed poikilotrophic or poikilophilic {poikilo 
= various; Gorbushina and Krumbein 1999). Poikilo-environments have prevailing 
hostile conditions for life (extremely low water potential, extreme temperatures, 
low nutrients, high levels of toxic substances), but the conditions may occasionally 
and sporadically change and become suitable for microbial activity and growth. 
The best examples of poikilotrophic organisms are rock-dwelling prokaryotes and 
fungi. Organisms living in deserts or arid fell-field soils with extremely low nutri- 
ents, precipitation, and highly variable temperatures can also be regarded as 
poikilotrophic. 

A more objective view of extreme environments is one based on the fact that 
there are specific physical and chemical limitations to cellular processes. These 
limitations are related to the characteristics of biomolecules and biochemical reac- 
tions and set the boundaries for cellular life. Extreme conditions can, according to 
this view, be defined as those near the limits for cell functioning, that is, limiting 
for enzyme activities or damaging to biomolecules (Rothschild and Mancinelli 
2001; Marion et al. 2003). The best example of a physical limit to life is the pres- 
ence of liquid water. Life is not possible without water, because it is the solvent 
necessary for all biochemical reactions. Other constraints are extreme conditions 
typical at the end of gradients, such as low and high temperature, low and high pH 
and E h , high salinity, high radiation doses, high concentrations of toxic compounds, 
and extremely low nutrient concentrations. Despite the physicochemical limits to 
biochemical processes and stability of biomolecules, the evolving micro-organisms 
have extended the boundaries for their life processes. 

Organisms living under extreme conditions are divided into different categories 
according to the nature of their adaptation (Table 2.1). They are classified as 
thermo- (high temperature), psychro- (low temperature), halo- (high salt), acido- or 
alkali- (extreme low or high pH), and xero- (low water activity). The suffix -phile 
is used for those that require the extreme condition for growth, and -troph or -toler- 
ant for those that tolerate the extreme condition. These designations are not exclu- 
sive, because two or more factors can be extreme in the same environment, as 
independent or interrelated conditions. The organisms may therefore belong to 
multiple categories, and, for example, be considered as both psychro- and xerophile. 

The extremophiles are adapted to and limited by very narrow sets of environmen- 
tal conditions, and they thrive in or require the extreme conditions. Extremotrophic 
or extremotolerant organisms can survive and proliferate under a wider set of envi- 
ronmental conditions. They tolerate extreme environments but normally grow better 
at moderate conditions. 

In ecology, the predominant growth strategy of organisms is often described as 
r- and K-strategy (Panikov 1999; van Elsas et al. 2006). Organisms with a predomi- 
nant K- strategy can live near the carrying capacity of the environment. They have 
relatively low growth rates, but compensate this by competitive advantages such as 



2 Microbial Life in Extreme Soils 



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18 V. Torsvik and L. 0vreas 

high affinity for substrates, low maintenance energy, the ability to uncouple growth 
from transport, and accumulation of storage polymers. In contrast, r-selected organ- 
isms have the potential of rapid proliferation and fast response to abundant and 
readily available substrates. A specific life strategy, designated L-strategy, is used 
to characterise organisms that are selected under unfavorable conditions and are 
highly tolerant or resistant to stress (Panikov 1999). This also includes micro-organisms 
with specific adaptation to manmade stress conditions. Thus, soils may be charac- 
terized as extreme which support the growth of micro-organisms that can tolerate 
anthropogenic disturbance and adverse conditions caused by high concentrations of 
pollutants and toxic compounds. 



2.2 Physicochemical Factors Limiting to Life 
2.2.1 Water 

Soil water is either adsorbed onto surfaces or present as free water in pores or films 
between soil particles. The soil water status is described by the water potential, 
which is a measure of the energy and forces that hold and move water in the soil. 
It relates to water activity (a w ) and is the difference in free energy (in pascals or Pa; 
energy per unit mass) between pure water and soil water. The main components of 
soil water potential are the matric potential (the energy with which soil water binds 
to solid surfaces or is retained in pores) and the osmotic potential (a function of 
dissolved salt concentrations; Vetterlein and Jahn 2004). Because these components 
reduce the free energy of water, the water potential is negative. This indicates the 
energy that organisms must exert to withdraw water from soil, and as the water 
potential decreases, water becomes less available and the stress level of organisms 
increases. 

Extreme soil water stress occurs periodically in most soils, even in climates 
with ample precipitation, where the water availability depends on soil composi- 
tion, rainfall drainage, and plant cover. Soil prokaryotes live in water films 
surrounding particles or inside water- filled pores, and are therefore very 
susceptible to water depletion. Soil fungi are normally more tolerant to water 
stress than prokaryotes. Furthermore, they can tolerate drought due to hyphal 
growth, which allows them to cross dried pores and obtain water from smaller 
pores where the water remains for longer periods (Killham 1994). Prokaryotic 
cells have a turgor pressure, with a concentration of solutes inside the cell being 
slightly higher than that outside. Changes in water activity in the environment are 
rapidly followed by a water flux across the semipermeable cell membrane from 
high to low water potential. This can cause swelling and lysis of the cells under 
hypotonic conditions or dehydration under hypertonic conditions (Kempf and 
Bremer 1998). Therefore the cell must maintain an intracellular water potential 
similar to that existing outside the cells. 



2 Microbial Life in Extreme Soils 19 

To maintain cell integrity at nonextreme temperatures (10-40°C) usually 
requires soil water potential above -4MPa (0.95 a w ) for most bacteria, and above 
-22MPa (0.86 a w ) for actinomycetes and most fungi. It is generally considered that 
the lower limit of water potential for life is -70MPa (0.60 a w ; Zvyagintsev et al. 
2005), but recently it has been demonstrated that spore germination and elongation 
of some actinomycetes can occur at water potential of -96MPa (0.50 a w ; 
Doroshenko et al. 2005; Zvyagintsev et al. 2005). Some organisms, for example 
lichens, can even survive on water vapor rather than liquid water. Inasmuch as cell 
damage cannot be repaired during desiccation, these organisms must exhibit an 
efficient repair system upon rehydration. 

Xerotolerant or xerophile micro-organisms are able to withstand water and salt 
stress because they can counterbalance a low water potential in the environment by 
accumulating highly soluble small molecules in the cytoplasm (Kempf and Bremer 
1998). The solutes can be inorganic salts or organic molecules (amino acids, polyols, 
carbohydrates, quaternary ammonium compounds). The accumulation of solutes 
results in decreased internal water potential. These molecules can influence and 
modulate specific enzyme activities, but do not inhibit the overall metabolism of the 
cells, and are therefore termed compatible solutes or osmoprotectants (see Section 
2.2.2). Some compatible solutes are constitutively produced, whereas others are 
induced. Osmoregulation is an energy-requiring process, but seems to be a general 
mechanism enabling soil micro-organisms to preserve the activity of intracellular 
enzymes under long-term and severe water stress. 

Other strategies that protect prokaryotes from desiccation are the production of 
extracellular polysaccharides which retain water (Wright et al. 2005). Formation of 
microaggregates of cells where elevated water activities are retained may further 
protect micro-organisms from desiccation. Actinomycetes are particularly well 
osmoregulated, as their cell membranes have restricted permeability and keep salt 
ions out and organic solutes inside the cells. Like fungi, they can differentiate into 
dormant cells that are resistant to drying (Dose et al. 2001). 



2.2.2 Salinity 

Salt or osmotic stress is closely related to water stress because solutes strongly 
affect the water activity. In contrast to water stress which occurs frequently in most 
terrestrial habitats, high salinity typically occurs in restricted habitats. Soils with 
high salinity are often characterized by highly uneven temporal and spatial water 
distribution (Brown 1976). Such fluctuations cause special stress for the microbes 
and reduce their ability to survive, because they need to respond rapidly to desicca- 
tion and adapt to high salt concentrations (see Chapter 5). 

Saline terrestrial habitats are typical for naturally arid regions with high evapo- 
ration rates. They may also be the result of pollution from mining activities or 
chemical and metallurgic industries. Most of the micro-organisms that inhabit 
saline soils are salt tolerant (halotolerant), but also halophilic micro-organisms 



20 V. Torsvik and L. 0vreas 

that require salt for maintaining their membrane integrity and enzyme stability 
and activity are present. Extremely halophilic prokaryotes can tolerate very low 
water potential, and grow well at -40Mpa (0.75a w , the value of saturated NaCl 
solution). This limit is determined by the solubility of salt rather than by the 
physiology of the cells (Brown 1976). As with the xerophile and xerotolerant 
organisms, the halophiles and halotolerant organisms accumulate a variety of small 
molecules in the cytoplasm (osmolytes or compatible solutes; see Section 2.2.2) to 
counteract the external osmotic pressure (Kempf and Bremer 1998; Roberts 2005). 
Generally, bacteria produce and accumulate compatible organic solutes that are 
zwitterions (e.g., proline, glycine betaine, ectoine, methylamines, and derivatives) 
or nonionic (such as polyols, carbohydrates, neutral peptides, and amino acids and 
derivatives). 

In archaea the osmolytes are often inorganic cations that are taken up by passive 
diffusion or by selective ion transport across the membrane. Many archaea have 
evolved negatively charged acid polypeptides that require cation counterions such 
as K + for proper protein folding and activity. Organic osmolytes that accumulate 
in archaea belong to the same types as for bacteria, but the majority of the solutes in 
archaea are anionic (due to negatively charged groups such as carboxyl, phosphate, 
and sulfate groups; Martin et al. 1999). In addition to their functions as osmotically 
active substances, the compatible solutes may function as chemical chaperones that 
protect proteins from denaturation and increase their activity. 



2.2.3 Temperature 

The temperature limits of life are related to the boiling and freezing points of water. 
However, many micro-organisms have developed mechanisms to extend the tem- 
perature ranges beyond the values for pure water and atmospheric pressure. At 
present, the temperature limitations for microbial activity are regarded as ranging 
from approximately -40°C to +130°C (Kashefi and Lovley 2003; Price and Sowers 
2004). Arctic micro-organisms are well adapted to an extremely cold climate and 
several authors have reported microbial activities at temperatures as low as -10 to 
-20°C (Panikov and Dedysh 2000; Bakermans et al. 2003; Jakosky et al. 2003; 
Callaghan et al. 2004; Gilichinsky et al. 2005; see Chapter 7). At subzero tempera- 
tures there can still be liquid water present in soils, as adsorbed water forms a thin 
liquid film on the surface of soil particles (hygroscopic water; Rivkina et al. 2000; 
Steven et al. 2006). 

Growth at low temperatures requires significant membrane alterations in order 
to maintain the fluidity necessary for nutrient transport across the membrane. 
The low temperature modifications involve less saturated and less branched 
membrane fatty acids. Below the minimum growth temperature the membrane 
becomes solid and transmembrane transportation stops. Life at subzero tempera- 
tures is also facilitated by accumulation of antifreeze compounds (high concen- 
trations of salts, hydrocarbons, or amino acids) in the cytoplasm. Archaea have 



2 Microbial Life in Extreme Soils 21 

many of the same mechanisms for adaptations to low temperatures as bacteria; 
these involve altered membrane composition (cold-adapted lipids) as well as 
cold-active proteins involved in fundamental cell functions (e.g., protein synthesis; 
Cavicchioli et al. 2000). 

In psychrophiles, the specific cold adaptation implies such drastic changes in the 
chemical composition of the cell that life outside of cold environments becomes 
impossible. For example, micro-organisms adapted to low temperatures have 
enzymes and ribosomes becoming unstable at temperatures 1-2°C above their 
optimum temperatures. Accordingly, the psychrophiles have optimum temperatures 
at or below 15°C and maximum temperatures below 20°C. The psychro trophic 
organisms can also grow at temperatures close to or even below 0°C, but their 
optimum temperature is above 15°C, and their maximum temperature can be as 
high as 30 to 40°C. Price and Sowers (2004) studied the temperature dependence 
of metabolic rates in different environments including permafrost. These authors 
distinguished three categories of metabolic rates: first, rates sufficiently high to 
allow growth; second, intermediate rates sufficient for maintenance of functions, 
but too low for growth; and third, basal rates sufficient for survival of cells and 
repair of damaged macromolecules, but otherwise permitting only cell dormancy. 
They did not observe any minimum temperature for metabolism, but at low 
temperatures the metabolic rate was extremely low. At elevated temperatures, 
micro-organisms from permafrost showed metabolic rates similar to those found in 
temperate soils. 

At the other extreme of the temperature range for life are the thermophiles and 
the thermotrophs. Thermophilic organisms cannot grow at temperatures below 
50°C, whereas the thermotrophs have a lower temperature limit (20-30°C). At their 
upper temperature limit, cells undergo instability and irreversible denaturation of 
their proteins and nucleic acids, and therefore the ability of these molecules to per- 
form their functions is lost. Thermal soils, with temperatures above 50°C, can be 
natural or manmade (see Chapters 8 and 14). Thermophilic micro-organisms have 
been isolated from natural thermal soils such as decomposing litter, volcanic, 
geothermal and tropical desert soils, and from manmade thermal soils such as com- 
post piles and coal refuse piles (Botero et al. 2004). 

Micro-organisms adapted to high temperatures have mechanisms for 
protecting their proteins and nucleic acids from irreversible denaturation. 
Biomolecules from such organisms are thermostable and remain active at tem- 
peratures that generally inactivate proteins, lipids, and nucleic acids in 
mesophilic organisms (Rothschild and Mancinelli 2001). In some proteins, the 
stabilization is caused by changes in amino acid residues that make the proteins 
more hydrophobic and increase the stability of subunit interactions (Singleton 
and Amelunxen 1973). The nucleic acids are also thermostabilized, for example 
as the result of interactions with histonelike proteins. At high temperatures, the 
membrane fatty acids acquire longer chains, and they become more saturated 
and more branched. Such changes in the membrane structure and composition 
lead to decreased membrane fluidity and consequently better thermostability 
(Pakchung et al. 2006). 



22 V. Torsvik and L. 0vreas 

2.2.4 pH 

Taken as a group, prokaryotes can live in environments with pH values ranging 
from below to 13 (Marion et al. 2003), although most prokaryotes grow at rela- 
tively narrow pH ranges close to neutrality. Extreme acidophiles grow in the pH 
range to 3. It seems that a general adaptation to extreme pH is to regulate the 
intracellular pH and keep it close to neutral. The bacterium Acidithiobacillus 
ferrooxidans (previously Thiobacillus ferrooxidans) lives in acidic environments 
with a pH of about 1 (see Chapter 10). However, its intracellular pH is around 5.5, 
which indicates an active mechanism for excluding protons. 

Whereas all known cytoplasmic enzymes have pH optima from pH 5 to pH 8, 
some enzymes found in the bacterial outer membrane tend to have low pH optima. 
Among archaea there are acidophiles that can grow at pH 0. Members of the cell 
wall-lacking archaeon Ferroplasma have been isolated from highly acidic envi- 
ronments associated with sulfide ores, solfatara fields, and the like (Golyshina and 
Timmis 2005). They can mobilize metals from sulfide ores and tolerate high con- 
centrations of heavy metals (Edwards et al. 2000; Baker- Austin et al. 2005). The 
extremely thermoacidophilic archaea Picrophilus torridus and P. oshimae were 
first isolated from solfataric Japanese soils. They have optimal growth rates at pH 
0.7 and 60°C (Schleper et al. 1995). In contrast to most other acidophilic 
micro-organisms, the intracellular pH is very low (pH 4.6), and P. torridus cannot 
grow above pH 4.0. A common feature of acidophile and acidotolerant 
micro-organisms is the presence of acidophilic lipids with cyclic rings and alkyl 
side chains in their fatty acids. Adaptation to acid environments is also enhanced 
by unusual tetraether lipids in the cell membrane (in Ferroplasma and Picrophilus) 
or lipopoly saccharides with a low content of fatty acids (in Acidithiobacillus). The 
tetraether lipids render the membrane of acidophilic archaea impermeable to 
protons, and a proton pump expels protons from the cytoplasm. High ratios of 
genes for proton-driven secondary transporters over ATP-consuming primary 
transporters indicate that the cells utilize the proton gradient for solute transport 
into the cell (Futterer et al. 2004). 

Extreme alkaliphilic micro-organisms have pH optimum between 9 and 1 1 , and 
do not grow near neutral pH. However, the cytoplasmic pH is at least two units lower 
than the external pH (Krulwich et al. 1998), which indicates that the cell membrane 
presents an efficient barrier to fluxes of OH" ions and that there is an efficient inward 
proton translocation system (e.g., Na + /H + or K + /H + antiporters, pro ton- translocating 
ATP synthase). pH in bulk soil is generally between 4.0 and 8.5 (Lynch 1979). 
Decomposition of plant litter tends to reduce the pH, and soil with accumulated 
organic matter is normally acid. Redox reactions also influence the soil pH. For 
example, oxidation of NH 4 + , S, and FeS 2 results in production of mineral acids such 
as HN0 3 and H 2 S0 4 , and thereby decreases the pH. Thus, it is apparent that prokary- 
otes exert a profound influence on their own environment. As a result of metabolic 
activity, the pH in microbial microhabitats can be at least 3-4 pH units lower than in 
the bulk soil. High pH is common in soils containing alkaline minerals, or occurs 
temporally in restricted zones, for instance due to the presence of animal excretions. 



2 Microbial Life in Extreme Soils 23 

Anaerobic reactions, such as the reduction of nitrate to N 2 and of sulfate to sulfide, 
also increase the pH. Recently alkaliphilic psychrotolerant bacteria were isolated 
from permafrost soil in the Qinghai-Tibet Plateau (Zhang et al. 2007). The colony- 
forming units of alkaliphilic bacteria in these soils varied between 10 2 and 10 5 cells 
g" 1 of dry soil. The isolates could grow at pH 6.5-10.5 with optimum pH of 9.0-9.5, 
and optimum growth temperatures of 10-1 5 °C. 



2.2.5 Radiation 

High doses of ionizing radiation and ultraviolet (UV) light are lethal to most 
microbes, although some of them can tolerate surprisingly high radiation doses (see 
Chapter 16). Normally high correlations are observed between tolerance towards 
radiation, desiccation, and DNA damaging chemicals (Shukla et al. 2007). 
Deinococcus radiodurans is the most radiation-resistant organism known and can 
survive doses of l,000J/m 2 UV-light, and more than 20kGy of y-radiation (1 Gy = 100 
Rad), which is approximately 4,000 times the dose that will kill a human (Battista 
1997; Marion et al. 2003; Rainey et al. 2005). This micro-organism is remarkably 
well adapted to extreme conditions as it can survive drought and lack of nutrients, 
in addition to extremely high radiation dosages (Battista 1997). This red spherical 
bacterium was discovered in 1957, in a can of ground meat that was spoiled despite 
having been sterilized by radiation. The bacterium is widely distributed and has 
been found in a variety of soil environments as well as in granite in Antarctic dry 
valleys. 

Dehydration and radiation cause very similar types of DNA damage. Resistance is 
conferred on Deinococcus by a particularly efficient system for DNA repair. 
Desiccation or high doses of radiation lead to massive double-strand breaks in DNA. 
Deinococcus has 4-10 copies of its chromosome (Battista 1997) and repairs the frag- 
ments by intrachromosomal recombination that reconstitute an intact chromosome in 
just a few hours (Minton and Daly 1995; Sale 2007). Prokaryotes and algae on 
surfaces of barren polar soil have adaptive strategies that allow them to avoid, or at 
least minimize UV injury. In this case, substances such as pigments and amino acids 
(e.g., melanoids, carotenoids, scytenomin, mycosporine-like amino acids) protect 
against the excessive light radiations and oxidative damage (Ehling-Schulz et al. 
1997; Bowker et al. 2002; Wright et al. 2005). 



2.2.6 Low Nutrients 

Environments characterized by extreme physical or chemical conditions, such as 
desert and permafrost soils, are often poor in organic and inorganic nutrients. Most 
of the indigenous micro-organisms in such environments are probably oligotrophs, 
which means they are adapted to low nutrient supply rates. However, high numbers 
of oligotrophic prokaryotes may also be found in bogs and other soils with high 



24 V. Torsvik and L. 0vreas 

amounts of organic matter. In such soils the major organic compounds are humic 
matter that is recalcitrant and not readily decomposable (Koch 2001; Fierer et al. 
2007). Bacteria in the phylum Acidobacteria are generally regarded as oligotrophic. 
They are especially abundant in soils with low resource availability and their abun- 
dance decreases after amendment with a readily available carbon source (Fierer et 
al. 2007). Prokaryotes that are adapted to grow in oligotrophic environments are 
normally K-selected (Bernard et al. 2007). They have low growth rates, but very 
efficient uptake systems with low half- saturation constants (down to nM levels) for 
uptake of organic substrates. This adaptation often results in their inability to grow 
under high nutrient levels. 

Other mechanisms for adaptation to low nutrient levels are the ability to use 
many different substrates simultaneously (Eichorst et al. 2007). In environments 
where nutrient supplies fluctuate, prokaryotes can store nutrients as intracellular 
polymers (e.g., polysaccharides, poly-P-hydroxybutyrate, polyphosphate). However, 
in constant oligotrophic environments, especially cold environments, the nutrient 
supply probably is too low to support any intracellular storage. It has been sug- 
gested that the organisms' affinity for substrates decreases at low temperature due 
to loss of membrane fluidity that impedes active transport, and that the minimum 
substrate concentration needed for growth therefore increases near the organisms' 
lower temperature limits (Wiebe et al. 1992). If liquid water is present, growth limi- 
tation by decreased temperature may be the result of reduced active uptake of 
nutrients, which eventually becomes so low that the cell's minimum maintenance 
requirements is no longer met (Nedwell 1999). 



2.2.7 Pollution 

Acid deposition has affected soil microbial communities and activities for some 
decades. This pollution is caused by acid precipitation, the result of nitrogen oxide 
(NO x ) and sulfur dioxide (S0 2 ) emitted into the atmosphere and oxidised to S0 4 2 ~ 
and N0 3 ". Despite the effort to reduce the primary sources of acid input, the effect 
is still apparent in many regions. The effect of acid deposition on soil ecosystems 
depends on the concentration of S0 4 2 ~ and N0 3 ", the amount of precipitation, and 
the buffering capacity of the soils (the cation exchange capacity through bases). 
Nitrogen and sulfur provided by acid rain may stimulate growth of some soil 
micro-organisms. On the other hand, even low-level but prolonged acid rain will 
result in soil acidification that may have adverse effects on soil bacteria, whereas the 
effect on fungi seems to be minor (Pennanen et al. 1998a; Baath and Anderson 2003). 

The effect of acid deposition can be direct or indirect. The lower pH and reduced 
concentrations of divalent cations (Ca 2+ , Mg 2+ ) can lead to mobilization and 
increased bioavailability of heavy metals and other toxic compounds (Francis 
1986). Acidification of soils may also reduce the solubility of organic matter and 
thereby reduce the substrate availability for microbes. Increased soil acidity does 
not seem to affect prokaryotic biomass to any extent, but rather to reduce prokaryotic 



2 Microbial Life in Extreme Soils 25 

growth rates and activity (Francis 1986; Pennanen et al. 1998b). Reduced activity 
of a number of soil enzymes, such as dehydrogenases, ureases, and phosphatases 
have been observed at significant pH reductions (Killham et al. 1983). The reduced 
microbial growth observed with increased acidity may indicate that more metabolic 
energy is used for maintenance rather than for biosynthesis of cell materials. It has 
been suggested that increased metabolic quotient (qC0 2 , the ratio of basal respira- 
tion to microbial biomass) indicates a shift in energy use from growth to mainte- 
nance, and that increased energy demand is a sensitive indicator of physiological 
adaptation to environmental stress (Post and Beeby 1996; Liao and Xie 2007). 

Soil can have naturally high concentrations of heavy metals as the result of 
weathering of parental material with high amounts of heavy metal minerals (e.g., 
mineral sulfides). Other sources are contaminations associated with mines and 
metal smelters, which have led to increased soil concentrations of heavy metals 
such as zinc, cadmium, copper, and lead. Sewage sludge may also contain heavy 
metals, and it has been demonstrated that long-term application of heavy metal 
containing sewage sludge to agricultural soils can have profound effects on the 
microbial diversity and community composition (Sandaa et al. 1999; Gans et al. 
2005). The effect of heavy metal toxicity depends on soil abiotic factors such as 
organic matter and clay content, divalent cation concentrations (cation exchange 
capacity), and pH (Giller et al. 1998). These factors influence complex formation 
and immobilization of heavy metals. 

Irrespectively of soil types, however, the relative toxicity of different metals 
seems to be the same, namely Cd > Cu > Zn > Pb (Baath 1989). In soil contami- 
nated for 40 years with high concentrations of Cr and Pb, the microbial biomass 
and activity was reduced and soil organic carbon accumulated (Shi et al. 2002). 
These results indicated that Pb presented greater stress to soil microbes than 
Cr. Soil micro-organisms vary widely in their tolerance to heavy metal contamina- 
tion, and the proportion of culturable resistant micro-organisms can range from 
10% to nearly 100%. The activity of enzymes in soil may serve as indicators for 
heavy metal contamination as there are generally high correlations between reduced 
enzyme activities (of, e.g., dehydrogenases, acid phosphatases, and ureases) and 
increased heavy metal contamination (Baath 1989). It has been reported that heavy 
metal contamination has a different effect on soil bacteria and fungi (Rajapaksha 
et al. 2004). Metal addition decreased bacterial activity whereas it increased fungal 
activity, and the increased fungal activity was found to persist in contaminated as 
compared to control soil after 35 days. The different effect of heavy metals was also 
demonstrated by an increase in the relative fungal/bacterial ratio (estimated using 
phospholipids fatty acid analysis) with increased metal concentrations. 

Mechanisms for metal resistance include stable complex binding (chelation) 
with organic ligands (extracellular or intracellular sequestering), transportation 
out of the cells, and biotransformation of the ions to less bioavailable or less toxic 
metal species. Genes for metal resistance (e.g., mercury resistance) are often har- 
bored on plasmids and can easily be disseminated through a population or a 
community in response to selection pressure associated with toxic metal exposure 
(Dr0nen et al. 1998). 



26 V. Torsvik and L. 0vreas 

Hydrocarbon contamination of soils caused by human activities increasingly 
occurs in all parts of the world. Petroleum is a rich carbon source and most of the 
hydrocarbon components are biodegradable by micro-organisms. The rate of deg- 
radation is normally rather low, because crude oil has low concentrations of phos- 
phorus and nitrogen, which do not allow extensive growth of indigenous 
hydrocarbon-degrading micro-organisms in petroleum-contaminated soils. 
However, growth can be stimulated by addition of phosphorus and nitrogen fertiliz- 
ers. In many extreme environments, hydrocarbon-polluted areas are found 
(Margesin and Schinner 2001). The success of bioremediation in such environ- 
ments depends on the presence of biodegrading microbes that are adapted to the 
prevailing environmental conditions. 

Pesticides are classified according to their primary target organisms, that is, her- 
bicides, fungicides, and insecticides (Johnsen et al. 2001). Normally the pesticides 
are very specific and restricted to a narrow range of target organisms. However, 
they can be modified in the environment and become toxic also to nontarget organ- 
isms. For instance, triazines, which normally target photosynthetic enzymes in C3 
plants, may be chlorinated in the triazine ring and thus become toxic to a wide 
range of organisms. The effect of pesticides on soil microbes depends on their bio- 
availability, which in turn is influenced by the crop being grown, as well as soil 
properties affecting the sorption and leaching of pesticides. The micro-organisms 
can develop resistance to the pesticides through their ability to decompose or trans- 
form them to less toxic compounds. 



2.3 Soil as Habitat for Micro-Organisms 

Soil has been defined as the upper weathered layer of the earth's crust, with a com- 
plex mixture of particulate materials derived from abiotic parent minerals, living 
biota, and particulate organic detritus and humic substances (Odum 1971). 
Formation of soil is the result of climate (temperature and moisture), parental 
material, time, topography, and organisms (Jenny 1994), and involves complex 
interactions of physical, chemical, and biological processes. Soil texture (the relative 
proportion of particles with different sizes) and mineral constituents depend on the 
parent material (rocks), and transportation by water, ice, and wind. Soil structure is 
the distribution of pores of various sizes that occur between soil particles. The pore 
sizes depend on the level of aggregation of soil particulate material, and the pores 
contain gases and water. 

The vegetation and soil biota affect soil development by weathering and control- 
ling organic matter accumulation and mineralization. The recognition of close 
interactions between soils and vegetation is reflected in the division of soils into 
major types, which are associated with climatic vegetation zones. Micro-organisms 
are able to modify and shape their physical and chemical environment. They 
dissolve and alter minerals derived from the parental material, contribute to and 
mineralize soil organic matter, and recycle nutrients. Microbes produce biopolymers 



2 Microbial Life in Extreme Soils 27 

(polysaccharides) as cell envelopes. Such polymers facilitate formation and 
stabilization of soil aggregates, and thereby improve the soil water-holding capacity. 
Together with colloid clay particles and humus, the polymers create complex 
structures with extensive surfaces, which adsorb minerals and organic molecules. 
Adsorption of proteins and nucleic acids to surfaces protects them from biodegra- 
dation and denaturation. Adsorbed DNA remains available for horizontal gene 
transfer by transformation of competent cells (Lorenz and Wackernagel 1994). The 
activity of extracellular enzymes is maintained or even increased by adsorption on 
minerals, whereas adsorption to humic substances can either maintain or decrease 
their activity (Nannipieri et al. 1990; Allison 2006). The adsorption to soil colloids may 
strongly reduce the availability of organic molecules as nutrients for micro-organisms, 
and contribute to soils being oligotrophic environments. 

Surfaces of soil minerals, especially clay colloids, can serve as catalyst for abi- 
otic chemical reactions. Clay particles are coated with metal oxides and hydroxides 
and have net electronegative charges. They can mediate electron transfer reactions 
and catalyse oxidation of phenols and polyphenols. They also contribute to humus 
formation by catalysing reactions such as deamination, polymerization, and con- 
densation of organic molecules. It has been suggested that microbial processes such 
as decomposition and mineralization of organic substances prevail under moderate 
conditions, whereas abiotic reactions are more dominant under harsh conditions 
where microbial activities are hampered (Huang 1990; Ruggiero et al. 1996). 

Soils are among the habitats that have been shown to support the highest abun- 
dance and diversity of micro-organisms. Soil habitats are distinguished from 
aquatic habitats by being much more complex and spatially heterogeneous. A char- 
acteristic feature is the wide range of steep physicochemical gradients (e.g., of sub- 
strate concentrations, redox potential, pH, available water) which may occur across 
short distances approaching the size of a soil aggregate. Thus, even an aggregate of 
a few mm can offer many different microenvironments that would collectively be 
colonized by different types of micro-organisms (Standing and Killham 2006). The 
size scale of microhabitats is typically a few jim for unicellular prokaryotes, but 
may be much larger for filamentous actinomycetes and fungi. Microhabitats for 
prokaryotes exist either within or between aggregates. Intra- aggregate habitats have 
typically small pores that are often water-filled and anaerobic, whereas inter-aggregate 
habitats are more frequently aerobic. However, the living conditions in these habitats 
can undergo considerable changes both in space and time, therefore soils are highly 
dynamic systems. 

The distribution, activity, and interactions (e.g., predation) of soil biota depend 
on soil properties such as texture, structure, and available nutrients and water. The 
growth conditions are normally most favorable on surfaces, and most (80-90%) of 
the soil micro-organisms are attached to surfaces (Hattori et al. 1997), often aided 
by extracellular biopolymers which stick to particles. However, surfaces also 
expose micro-organisms to the highest risks of desiccation and predation. Specific 
soil habitats such as organic litter aggregates, biofilms, rhizosphere, and animal 
droppings, are rich in readily available organic nutrients and can support very high 
microbial activities. The bulk soil on the other hand often contains low levels of 



28 V. Torsvik and L. 0vreas 

easily decomposable substrates, and most of the organic matter is refractory. Thus 
the distribution of biomass and activities of soil microbes is generally very patchy, 
and the space that is occupied by micro-organisms may be less than 5% of the 
overall space in soil (Nannipieri et al. 2003). 

Moderate soils with no stress factors are characterized by a high microbial abun- 
dance (10 9 -10 10 prokaryotes g" 1 soil dry weight) and high genetic, phylogenetic, and 
functional diversity of microbial communities (Giller et al. 1997; Torsvik and 
0vreas 2006). Micro-organisms are by far the most active and functionally diverse 
component of the soil biota. It has been estimated that 80-90% of the soil processes 
are mediated by the microbiota, including prokaryotes and fungi. Generally, about 
one third of the organic carbon added to temperate soils is transformed to humus 
and microbial biomass, whereas about two thirds of the carbon is respired to C0 2 
by micro-organisms (Stotzky 1997). 

Interestingly, the deep subsurface terrestrial environments, which can extend for 
several hundred meters below the soil surface, have been proven to sustain ample 
microbial biomasses. Although the cell numbers are much lower than in the surface 
soil, a variety of micro-organisms, primarily prokaryotes, is present in deep subsur- 
face soils. For example, in samples collected aseptically from bore holes drilled 
down to 300 m, a diverse array of micro-organisms has been found. These organ- 
isms most likely have access to organic nutrients present in the groundwater perco- 
lating down the subsurface material and flowing through their habitat. Studies on 
the microbial ecology of deep basalt aquifers have shown that both chemoorgano- 
trophic and chemolithotrophic prokaryotes are present, but that the chemolitho- 
trophs are dominating in these environments (Stevens and McKinley 1995). 



2.4 Extreme Soils 

Extreme soil microbiology deals with micro-organisms adapted to extreme or 
stressful soil conditions. Soil properties are determined by the parental material 
(geological properties), climate, and biota, and are influenced by anthropogenic 
activities. Odum (1971) divided soils in two categories, those which are mainly 
controlled by climate and vegetation types, and those which are mainly controlled 
by parent materials or other pedological or environmental factors (topography, 
drainage, pollution, etc.). These controlling factors can strengthen each other. In 
regions with extreme climate, minor differences in edaphic factors may create large 
differences in the structure and activity of soil microbiota. 

In many temperate climate zones, soil water and/or temperature stress occurs 
periodically. Also nutrient stress occurs periodically in many soils, and this will 
influence the soil microbiota so that organisms adapted to periodic stress become 
dominant. In some areas, wide fluctuations in environmental conditions occur. It 
has been reported that in Antarctic desert soil the temperature could change from 
-15°C to nearly 30°C in three hours (Cowan and Tow 2004). For the micro-organisms 
to survive freeze-thaw cycles and sudden differences of more than 40°C, very 



2 Microbial Life in Extreme Soils 29 

specialized adaptations are required. Thus, in soil where a stress situation is 
maintained over an extended time period, the microbiota will develop specialized 
adaptations and life strategies that differentiate them from microbiota in nonex- 
treme environments. However, such specialization may correspond to a tradeoff 
between life under adverse and harsh situations and loss of adaptability. Indeed, 
extremophiles are often not able to adapt to less extreme conditions, and do not 
compete effectively under moderate conditions. 

Soil microbial communities under nonextreme and relatively stable environmen- 
tal conditions are characterized as functionally redundant. Moderate environmental 
stress and perturbations seem to have little impact on overall soil processes such as 
respiration and mineralisation, although the microbial community structure can be 
profoundly changed. This is explained by the insurance hypothesis (Yachi and 
Loreau 1999), which states that in an ecosystem there are many different popula- 
tions which can perform the same function, so that when some micro-organisms 
disappear others proliferate and take over the function (Giller et al. 1997). Microbial 
communities in extreme environments, especially those with fluctuating conditions, 
often comprise some numerically dominant species. In such environments, ecologi- 
cal processes may be more sensitive to changes in diversity imposed by additional 
stress factors. Lack of functional redundancy in extreme soils is illustrated by the 
observation that, in these environments, microbiota plays an increasing role at all 
trophic levels. For example, cyanobacteria and microalgae contribute significantly 
to primary production when the conditions become so harsh that higher plants can 
no longer grow. 

Two types of extreme soils are dominant on Earth, namely desert and tundra 
soils. Typical of these biomes is that vegetation is sparse and consists mainly of low 
vegetation, with no trees being present. 



2. 4. 1 Desert Soils 

Water is an overall limiting factor in terrestrial ecosystems, and within a specific 
climate zone the annual net primary production correlates well with annual precipi- 
tation. Deserts occur in regions having less than 250 mm of rainfall per year (Odum 
1971). Arid areas, that can be either extremely hot or cold, cover more than 30% of 
Earth's terrestrial surface (Rainey et al. 2005). Dry soil ecosystems are characterized 
by spatial patterns and high spatial heterogeneity. In temperate deserts, spatial 
variability is strongly influenced by vegetation, whereas in polar deserts, which 
often lack vascular plants, physical processes control the spatial variation in soil 
properties. An example is the formation of frost fissure patterns. 

The micro-organisms living in such environments have to deal with unfavorable 
life conditions such as absence of water, high or low temperatures, and lack of 
nutrients. The most extreme deserts are found in Antarctica (Ross Desert, Dry 
Valleys), in northern Chile (Atacama Desert ; see Chapter 6), and in central Sahara 
where there is virtually no rainfall. The low precipitation is caused by high 



30 V. Torsvik and L. 0vreas 

subtropical atmospheric pressure (Sahara), position in rain shadow areas (Chile), 
high altitude (Tibet and Gobi), or latitude (Antarctic Dry Valley). In hot deserts, 
most of the matric water evaporates during the day and micro-organisms obtain 
moisture by absorbing dew water during cool nights. The cold deserts in Antarctica 
suffer from extreme temperature in addition to extreme water stress, although in 
some areas and during restricted periods water can come from melting snow. 
The air temperature is -10°C on average during the summer and down to -55 °C 
during winter, and there are often strong winds, which are responsible for high 
sublimation rates. 

In the past, doubts were raised if any organism could proliferate under such cli- 
matic conditions, but micro-organisms have been isolated from even the harshest 
desert environments. In deserts, the microbiota often inhabit pores in sandstones or 
they tend to form biological soil crusts. In the Ross Desert, an Antarctic cold desert, 
cryptoendolithic micro-organisms grow in the near- surface layer of porous sand- 
stone rocks, where the microclimate is less hostile. They transform and mobilize 
iron compounds, and depend on the unsteady interactions between biological and 
environmental factors for survival. If the balance between these factors changes and 
becomes unfavorable, they will die but leave behind trace fossils and a characteris- 
tic iron-leaching pattern caused by their activity (Friedmann and Weed 1987). In 
the most extreme cold deserts, conditions suitable for microbial metabolism may 
occur only 2-10 days per year. 

The crust communities are composed of prokaryotes, fungi, microalgae, and 
lichens. They are extremely important in desert ecosystems as they form stable 
soil aggregates with increased water retention responsible for functions such as 
primary production, nitrogen fixation, and nutrient cycling. Lichens are espe- 
cially well adapted to extreme conditions, as they can withstand desiccation for 
long periods. Under cold conditions, the lichen algae keep the water in their 
cytoplasm in liquid form by producing and accumulating polyol intracellular 
solutes. The desiccation tolerance characteristic for desert micro-organisms is 
often correlated with salinity tolerance, extreme oligotrophy, and radiotolerance. 
Chanal et al. (2006) analysed microbial diversity in the Tataouine sand dunes in 
south Tunisia. The climate at this site is arid with a high seasonal variation in 
precipitation. The mean annual rainfall is 115 mm, and there is almost no pre- 
cipitation in summer. Despite these unfavorable conditions, an unexpectedly 
high diversity of micro-organisms was revealed. The community contained a 
broad spectrum of micro-organisms, with 16S rRNA sequences affiliated with 
11 bacterial divisions and some archaeal lineages. After irradiation of this soil 
with 15kGy, radiotolerant organisms affiliated with Bacillus, Deinococcus/ 
Thermus, and the Alphaproteobacteria could be isolated. In fact, many of the 
environments from which radioresistant organisms have been isolated are 
extremely dry, and many of these isolates are also desiccation-resistant (Rainey 
et al. 2005; Chanal et al. 2006). 

In deserts with the most extreme dry habitats, micro-organisms can be totally 
dried out, and in arctic deserts they may actually be freeze-dried. The best strategy for 
micro-organisms to survive in such extreme environments may be to completely 



2 Microbial Life in Extreme Soils 3 1 

abolish their metabolism during the most unfavorable time period, and switch into 
a dormant state until the conditions improve. Therefore many of them have resting 
stages or spores (Barak et al. 2005). 



2.4.2 Tundra Soils 

Climate has an overriding effect on species diversity on a global scale and biodiver- 
sity generally decreases with increased latitudes and altitudes. For eukaryotes, this 
trend is seen in polar regions both in terms of number of species and growth forms. 
However, on smaller spatial scales, biodiversity may not be any lower in Arctic 
tundra than in temperate soils. Permafrost represents approximately 26% of terres- 
trial soil ecosystems and can extend hundreds or even thousands of meters down 
into the subsurface (Steven et al. 2006). The permafrost environment is considered 
extreme because indigenous micro-organisms must survive long-term exposure to 
subzero temperatures and withstand background radiation. Low temperature and a 
short growing season (approximately 60 days) characterize extreme tundra and 
high altitude fell-field soils. Here such soils are considered and described together 
as extreme environments. 

On a global scale, most of the tundra consists of Arctic wetlands covered 
by vegetation. However, the most extreme high Arctic tundra offers bare soil 
without vegetation except for sparse areas of lichens, sedges, and grasses. In the 
high Arctic we find permafrost soils, where the ground is permanently frozen 
except for a few dm of active layer during the growth season (see Chapters 7 and 
12). Characteristic in permafrost soils are the ice-wedge polygon structures. 
These are topographic features formed by a network of ice-wedges, with either a 
depressed central area caused by thawing of the ice-rich permafrost in the centre, 
or a relatively elevated central area due to melting of the surrounding ice-wedges 
(see Chapter 7). Alpine fell-field tundra occurs in high mountains in temperate 
zones, and such tundra soils do not have permafrost. 

In some areas such as the Antarctic deserts, several harsh environmental 
factors interact, such as low temperature, low annual precipitation, and strong 
desiccating winds. The Antarctic Dry Valleys are regarded as the coldest and 
driest place on Earth. The precipitation is only a few millimetres a year and 
occurs mainly as snow. As most of this snow is blown away, the potential 
evaporation exceeds precipitation. These regions are further characterised by a 
long period of winter darkness and low temperatures, followed by a very short 
summer with 24 h daily light for a few weeks, and even then the temperature 
rarely exceeds 0°C. Organisms in these environments must therefore tolerate 
long periods of desiccation and dormancy, and a common opinion has been 
that the microbial biomass is very low in these soils (Horowitz et al. 1972; 
Virginia and Wall 1999; Smith et al. 2006). Recent investigations suggest that 
the biomass is several orders of magnitude higher than previously recognized 
(Cowan et al. 2002). 



32 V. Torsvik and L. 0vreas 

In polar tundra areas there may also be profound microclimate differences. One 
factor which exerts a major influence on soil temperature is the snow depth. In 
Siberia and Alaska, it has been observed that, whereas exposed areas with low snow 
cover had soil temperatures down to -30 to -40°C, soils under the snow cover had 
temperatures around -5 to -10°C. During the active summer season the diurnal 
temperature fluctuations in the upper soil layers can vary considerably over short 
time periods. The amplitude of such fluctuations is influenced by the soil water 
content and vegetation cover, but in dry barren mineral soils temperatures can vary 
by more than 20°C, sometimes by nearly 40°C, with minima below 0°C. As a result 
of such abrupt temperature changes, freeze-thaw cycles occur that can be lethal to 
soil organisms. The organisms have therefore developed mechanisms that allow 
them to survive repeated freeze-thaw cycles. 

Survival of adapted microbes depends on their hydration state, their compatible 
solute content, and their ability to switch metabolism to cryoprotectant synthesis. In 
some arid mineral soils, the micro-organisms are also subjected to osmotic stress due 
to accumulated salts. However, the presence of salt may result in water remaining 
liquid in cold environments, and active microbes can exist in thin films of liquid 
water present in permafrost or in permafrost brine lenses, called cryopegs, at below 
freezing temperatures (Gilichinsky et al. 2003). Cryopegs are layers of unfrozen 
ground that are perennially cryotic (forming part of the permafrost), but in which 
freezing is prevented by freezing-point depression due to high concentrations of dis- 
solved substances in the pore water. An unfrozen cryopeg is entirely surrounded by 
frozen ground (Gilichinsky et al. 2005). Such habitats allow for microbial growth at 
-10°C and metabolic activity at -20°C and even lower (Bakermans et al. 2003). 



2.5 Microbial Diversity and Community Structure 
in Extreme Soils 

The term microbial diversity describes different aspects of complexity and varia- 
bility within microbial populations and communities. This comprises genetic 
variability within taxons (species), variability in community composition, complexity 
of interactions, trophic levels, and number of guilds, this latter parameter defining the 
functional diversity. Diversity is expressed in different ways: as inventories of 
taxonomic groups or as single numbers (diversity indices), which are based on the 
number of taxons or OTUs (operational taxonomic units). Diversity may also be rep- 
resented as phylogenetic trees, or appreciated from the number of functional guilds. 
In moderate and stable environments, soil microbial communities will normally 
develop into complex systems with high phylogenetic and functional diversities. 
Therefore, such communities are among the most difficult to characterize pheno- 
typically and genetically. In addition, huge and coherent discrepancies between the 
total and cultivable cell numbers in natural environments has led to the introduction 
of "the great plate count anomaly" concept (Staley and Konopka 1985). This means 
that diversity measurements based on cultured micro-organisms are restricted to a 



2 Microbial Life in Extreme Soils 



33 



subset of 1% or less of the community members (Torsvik et al. 1990; Ward et al. 
1990) and applying culture-dependent methods will only reveal information about 
the very small fraction of micro-organisms able to grow under the given conditions 
(S0rheim et al. 1989). 

Molecular methods and direct in situ studies circumvent the selective and biased 
culturing step and allow both cultured and noncultured members of a community 
to be surveyed (Pace et al. 1986; Torsvik et al. 1998). Some molecular methods 
allow for an in situ detection of prokaryotes in more or less intact soil samples 
whereas other methods require effective separation of cells from soil particles. 
Analysis of total DNA extracted directly from a community generates information 
derived from all the community members, and provides estimates of the microbial 
diversity and a comprehensive picture of soil microbial community composition 
(Fig. 2.1). The information contained in nucleic acids can be used to address diver- 
sity at different levels from the entire microbial community and populations to 
within species levels. A schematic overview of various methods used to obtain such 
information is given in Fig. 2.2 and in 0vreas (2000). 

The total genetic diversity can be estimated by measuring the reassociation rate 
of community DNA (Britten and Kohne 1968; Torsvik et al. 1990), which is a 
low-resolution method that allows analyses of broad-scale differences in microbial 
communities (Torsvik and 0vreas 2006). 



Soil sample 



Cell based studies 





Direct in situ studies 



Cultivation 
dependent methods 





Nucleic acid extraction 

i 

Molecular methods 



tf 



Fig. 2.1 Schematic drawing showing the overall approaches available for measuring bacterial 
diversity in soil 






V. Torsvik and L. 0vreas 



Metagenomic 
cloning 



i 



Extraction of community 
nucleic acids (DNA/RNA) 

CD O 
QOCD 



PCR 



RT-PCR 



Metagenomic library 







Community rRNA genes/ 
/ functional marker genes 

4 I 

Shotgun 



cloning 



Fingerprinting analyses; 
DGGE,ARISA,T-RFLP,S5CP 



Genome sequencing 




i 



i 



Database search and 
sequence annotation 




DN A sequencing 






I 



Metagenome 
sequence 



Database search, 

construction of phylogenetic 

trees and probe design 




Melting profiles/ 
GC determination 




! 

Reassociation 

Fv] 



I 



Estimation of Ecoli 
genome equivalents 



Fig. 2.2 Schematic drawing of the basic principles of some molecular methods 



The metagenome can be regarded as the sum of all the microbial genomes in a 
given sample. Therefore, the metagenome approach represents a different whole 
community DNA-based analysis. It circumvents the cultivation anomaly as well as 
the PCR biases by cloning and sequencing genes directly from the environmental 



2 Microbial Life in Extreme Soils 35 

DNA. This method involves construction of complex community libraries by direct 
cloning of large genomic DNA fragments (40-80 kb) from environmental samples 
into fosmid or BAC (bacterial artificial chromosome) vectors. The challenge of this 
application lies in the ability to extract DNA of high molecular weight and high 
purity. The metagenome approach can be used to generate information on the 
potential functioning of individual microbial species in soil environments in order 
to study the broader role of micro-organisms in the ecosystem (Rondon et al. 2000; 
Tringe et al. 2005). 

The most common approach for assessing microbial diversity is to use polymer- 
ase chain reaction (PCR) to amplify 16S rRNA genes (rDNA) from the community 
(Pace et al. 1986). The amplified genes can then be cloned, and clones can be iden- 
tified by DNA sequencing, which are then amenable to comparative analyses. 
Cloning and sequencing approaches are time-consuming and labor-intensive for 
routine analysis of large sample sets. To screen for changes in time and space, com- 
munity fingerprinting techniques such as denaturing gradient gel electrophoresis 
(DGGE), terminal restriction fragment length polymorphism (T-RFLP), automated 
ribosomal intergenic spacer region analysis (ARISA), and single strand conforma- 
tional polymorphism analysis (SSCP) of PCR amplified 16S rRNA genes are 
frequently used (Muyzer et al. 1993; Macnaughton et al. 1999a,b; Kozdroj and van 
Elsas 2001; Kuske et al. 2002; Hoj et al. 2005, 2006; Nakatsu et al. 2005; Neufeld 
and Mohn 2005; Becker et al. 2006; Joynt et al. 2006; Loisel et al. 2006; Hewson 
et al. 2007). 

All these methods provide information about the numerically dominant commu- 
nity members, and the motivation for choosing one particular method instead of 
another lies in the expertise and equipment available in various laboratories. 
Furthermore, the phylogenetic affiliation of the numerically dominant organisms 
can be assessed by subsequent sequence analysis of, for instance, DGGE- separated 
PCR products. Such rDNA-based fingerprinting and cloning approaches offer 
higher resolution than DNA reassociation analysis, and have led to the discovery of 
several new prokaryotic taxa, even some entirely new divisions (Hugenholtz et al. 
1998, 2001). Many studies have showed that most of the 16S rRNA sequences 
obtained from a given soil sample are unique (Hugenholtz et al. 2001; Smith et al. 
2006). Due to the complexity of soil communities and the efforts required for the 
cloning and sequencing-based approach, only a limited number of soil environ- 
ments have been surveyed using such methods, and our understanding of the extent 
of microbial diversity in soils is still very limited. More detailed descriptions of 
molecular methods for analysing microbial communities are provided in recent 
review papers (Johnsen et al. 2001; Prosser 2002; Lynch et al. 2004; van Elsas 
et al. 2006). 

Regardless of their limitations, the culture-independent molecular methods 
have greatly expanded our view of extreme soils as habitats for micro-organisms. 
Several investigations have addressed the effect of extreme conditions created by 
human activities and pollution on microbial communities. Microbial communities 
in soils treated for many years with heavy metal-contaminated sewage sludge 
were investigated with respect to diversity and composition (Sandaa et al. 1999). 



36 V. Torsvik and L. 0vreas 

The control soil was amended with "uncontaminated" sewage sludge, whereas 
the contaminated soils received sewage sludge with two different levels of heavy 
metal concentrations (resulting in low and high levels of metal contamination). 
The total genetic diversity in microbial communities in unpolluted soil was high. 
In this case, the complexity of the community genome corresponded to approxi- 
mately 9,800 different bacterial genomes with an average E. coli genome size. The 
diversity of the metal-polluted soils was reduced and depended on the level of 
pollution. The complexity of DNA isolated from the soils with low and high lev- 
els of metal pollution corresponded to a diversity of approximately 4,600 and 
1,500 E. coli genomes, respectively. Thus, it seems that the genetic diversity can 
be an indicator of environmental stress caused by pollution. 

It was further observed that environmental stress induces profound changes in 
the community structure. Pollution and perturbation lead both to reduced species 
richness and evenness, as some species become numerically dominant. It can be 
concluded that, under extreme stress conditions or strong pollution, microbial 
diversity may be reduced, and microbial community structure changed to the 
extent that functioning of this community is altered (Giller et al. 1997; Griffiths 
et al. 2004). 

Both DNA reassociation and clone library analysis suggest that the overall 
prokaryotic diversity in pristine Arctic tundra soils can be very high (see Chapter 7), 
even higher than in soils from temperate regions (0vreas et al. 2004; Neufeld and 
Mohn 2005). The prokaryotic communities in tundra soils have representatives of 
the same phylogenetic divisions as found in soils at lower latitudes (Cowan et al. 
2002; Smith et al. 2006). They also carry out the same microbial processes as in 
temperate soils although at slower rates. 

The increased human activities in polar regions depend on petroleum hydrocar- 
bon for power generation and transportation. As a consequence of this and the 
exploitation of oil field reservoirs in the Arctic, increased oil pollution has become 
a significant problem in these cold environments (Aislabie et al. 2006; see Chapter 12). 
Several studies of hydrocarbon-contaminated polar soils indicate that hydrocarbon 
degraders are widely distributed in polar soils and that oil spill will result in marked 
enrichment of these micro-organisms (Atlas 1986; Aislabie et al. 2004, 2006; 
Sunde 2005). In comparative studies of pristine and oil-polluted Arctic and 
Antarctic tundra, significant shifts in the microbial diversity and community com- 
position have been observed as a result of oil contamination. Disturbed tundra soil 
had lower microbial diversity than pristine soils, and in the polluted soils some 
populations were very predominant (Neufeld and Mohn 2005; Saul et al. 2005; 
Smith et al. 2006). 

An investigation from Arctic tundra at Svalbard, Norway, showed that the pro- 
portion of clones with sequence similarities to cultured bacteria was much higher 
in polluted (36%) than in pristine soil (6%). Even then, the phylogenetic groups that 
were most abundant in pristine tundra soil have so far only been represented in the 
databases by a limited number of sequences from cultured organisms (Yndestad 
2004). These, as well as clone library data from Antarctic pristine tundra soils, 
indicate that most of the sequences are derived from unknown and uncultured 



2 Microbial Life in Extreme Soils 37 

micro-organisms, and may represent new and undescribed taxa. Enrichment 
cultures with three different oil types incubated at 4°C demonstrated that different 
oils promoted the establishment of different communities. In these experiments, the 
efficient oil-degrading organisms showed phylogenetic affiliation to well-known 
hydrocarbon-degrading organisms within the Proteobacteria and the Gram positive 
bacteria (Sunde 2005). In oil-contaminated soil, biodegradation of petroleum 
hydrocarbons by indigenous cold-adapted microbial populations at low tempera- 
tures has been observed (Whyte et al. 1999, 2001; Rike et al. 2003; Sunde 2005), 
but the in situ rates of degradation were low. Therefore, the activity of the 
indigenous hydrocarbon-degrading microbes is limited in cold soil, most likely by 
a combination of unfavorable conditions including low temperature and moisture, 
nutrient limitation, alkalinity, and potentially inhibitory hydrocarbons. 



2.6 Conclusions: The Significance of Studying 
Extremophiles in Soil 

Extreme soils have highly selective physicochemical properties and many of them 
have low microbial diversity relative to nonextreme soils. Therefore, they can serve 
as model systems for exploring fundamental ecological principles such as the rela- 
tionships between diversity and activity of micro-organisms and soil environmental 
conditions (Smith et al. 2006). Furthermore, studies of microbial community com- 
position and functions in extreme soils may be of great value for applications in 
environmental cleanup, pollution prevention, or energy production. 

Improved knowledge about extreme ecosystems will lead to important advances 
in the understanding of microbial adaptation mechanisms, and facilitate the design 
of biotechnological applications for enzymes and other compounds adapted to 
function under extreme physicochemical conditions. In addition, extreme prokaryo- 
tes may be interesting for bioprospecting, as they can be expected to contain a 
number of bioactive compounds potentially useful in medicine as well as in the 
pharmaceutical and environmental industries. 

Defining the limiting conditions for life on our planet can aid us in speculation 
on comparable limits in the universe. Extreme environments on Earth may resem- 
ble those that exist on other planets and moons. Thus, investigation of the most 
challenging environments on Earth, can give us some clues as to under which 
conditions we can expect to find life on other planets (see Chapters 6, 7, and 10). 
It can also provide some hints of what to search for when looking for signs of 
extraterrestrial life (see Chapter 11). The study of microbes in extreme soils is 
therefore highly relevant for astrobiology. It will advance our understanding, at 
the molecular and physiological levels, of specializations and adaptations 
required for the maintenance and proliferation of remote and as yet unrecognized 
forms of life. 

Acknowledgements We thank Beate Helle for technical assistance with the figures. 



38 V. Torsvik and L. 0vreas 

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Chapter 3 

Extreme Views on Prokaryote Evolution 



Patrice Dion 



Wilt thou not view, then, the truth, in my mirror so clearly 
depicted? 

- Goethe 

3.1 Introduction 

Not two soils are identical. Not two bacteria are identical either, with perhaps even not two 
daughter cells as they emerge from symmetric division being rigorously the same (Stewart 
et al. 2005). However, it might be easier to draw generalizations from observations of 
Escherichia coli or Caulobacter crescentus than from soil studies. This might explain why 
the bacterial cell has been deemed a more popular object of theoretical reflections than has 
the soil environment, although remarkable concepts on soil biology have emerged (Wardle 
et al. 2004; Young and Crawford 2004). This being said, extreme soils may appear as a 
particularly fruitful ground for those of us who feel seduced by the "idea of the soil" and 
who wish to venture on such a (admittedly slippery) terrain. 

Life constantly reinvents itself, under new aspects that owe much to ancient ones, 
to the point that nothing, at least microbial, can be said to have ever disappeared: the 
author is aware that this opinion may hold only if emphasis is placed on the modular 
construction of prokaryotic cells. In this sense, the last universal common ancestor or 
rather perhaps a set of basic processes acting on communally evolving primitive cells 
(Woese 2002), is still among us in a transformed and diversified form, and having 
seeded among its progeny many clues of its ancient organization. This apparent per- 
sistence of functional attributes over evolutionary time has been termed the "fecun- 
dity of function principle" (Staley 1997). Anecdotally, it might also be remarked that 
even the smallpox virus is still preserved somewhere behind well-locked doors. 

Life on Earth has been proposed to have originated in a hot (Di Giulio 2003; 
Schwartzman and Lineweaver 2004) or a cold (Price 2007) environment. As a rec- 
onciling alternative to these opposing possibilities, it has also been suggested that 



Patrice Dion 

Departement de phytologie, Pavilion Charles-Eugene Marchand, 1030, avenue de la Medecine, 

Universite Laval, Quebec (Quebec), Canada G1V 0A6 

e-mail: patrice.dion@plg.ulaval.ca 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 45 

© Springer- Verlag Berlin Heidelberg 2008 



46 P. Dion 

the earliest organisms might have been mesophiles, soon to be replaced by ther- 
mophiles which would have been advantaged, in particular, by their greater 
resistance to various stresses (Mas et al. 2003). Indeed, various aspects of primitive 
environments might have been rude by human standards. Now we see that climates 
and environments are changing, with soils becoming hotter, colder, dryer, more 
saline or toxic, the risk of nuclear accidents or fire notwithstanding. So the 
examination of microbial life reactions to these changes (Staley 1997) is timely. 



3.2 Scope and Limitations of This Review 

This review attempts to describe extreme soils as two-component environments, and to 
examine the corresponding bacterial response. One must obviously be guarded against 
generalizations such as those that are made here, as the genetic response to selection 
varies among different bacterial species and even among different regions of the 
genome of a given organism (Van Belkum et al. 1998). As a second word of caution, it 
must be stressed that not considered in this review is the role of horizontal gene transfer 
in microbial adaptation to extreme soil conditions, and especially the possibility that 
mutators would cooperate with nonmutator variants to generate adapted strains with 
preserved essential genes (Loewe 2004). In fact, horizontal gene transfer plays a major 
role in bacterial evolution (Goldenfeld and Woese 2007), and its contribution to the life 
and metamorphoses of extreme soil micro-organisms would be well worth investigating. 
Finally, the author is aware that soils are fluctuating environments, where stochastic 
phenotypic changes (Kussell and Leibler 2005) and phase variations (Thattai and van 
Oudenaarden 2004) might occur, and where persisters (Kussell et al. 2005) might arise, 
influencing evolutionary responses. However, a static point of view is adopted here, in 
the hope of designing a general framework sufficiently broad and solid to later accom- 
modate evolutionary changes accompanying environmental fluctuations and the hori- 
zontal transmission of discrete, perhaps modular, characters. 

Prokaryotic evolution has given rise to deep divisions reflecting ancient divergences in 
cell structure and function (Woese 1987), but it has also produced comparatively minute 
differences within a species, subspecies, or clone (Schloter et al. 2000). The relationships 
between the mechanisms of macroevolution and microevolution are not elucidated, and it 
has been suggested that macroevolutionary events cannot be deduced from a simple 
extrapolation of microevolutionary changes (Erwin 2000). However, macroevolution and 
microevolution are both based on environmental and clade constraints (Moore and Woods 
2006), implying that there need not be mechanistic differences between macroevolution- 
ary and microevolutionary changes. Specifically, punctuated evolution may occur in 
clonal bacterial populations by simple mechanisms such as selection of rare mutations 
with a large effect (Elena et al. 1996). Many of these mutations are likely to be pleiotropic, 
altering global regulators (Zinser and Kolter 2004). Hence, it appears that, for the sake of 
simplicity at least, major evolutionary changes and minor adjustments, which would both 
be expected to occur in micro-organisms adapting to extreme soil conditions, can be 
accommodated within the same evolutionary framework. 



3 Extreme Views on Prokaryote Evolution 47 

3.3 Selection as It Occurs in the Laboratory 
and in the Real World 

Experiments on bacterial evolution are conducted under laboratory conditions that 
differ markedly from those that prevail in extreme soils or other environments. 
Evolutionary changes are provoked in the laboratory by applying unique and 
discriminating selective pressures. Typically, bacteria are experimentally challenged 
for the utilization of a particular carbon source, or for phage or antibiotic resistance. 
In most cases, the experimental design calls for an all or none response, such as life 
or death, growth or no growth. However, some experimental systems have led to 
the observation of small fitness gains and to an elucidation of the mutations 
involved (e.g., see Cooper et al. 2001). 

A successful response to experimental selective conditions generally involves 
the modification of a single cell component, such as a catabolic enzyme or a phage 
receptor (Cairns et al. 1988). On the contrary, environmental challenges to bacteria 
are multiple and often associate to build global physiological constraints. Bacteria 
may gain advantage from the utilization of a substrate, not as the sole, but as an 
additional carbon source. They may benefit from resistance to a particular 
bacteriophage, while remaining sensitive to other viruses and prey to protozoa. 
Even a slight increase in growth rate, for example resulting from adaptation to 
physical conditions or chemical resistance, may contribute usefully to environmental 
success. Thus, the extent to which the results of laboratory studies on bacterial 
evolution can be extrapolated to natural systems calls for careful evaluation. 

At least two aspects of this problem ought to be considered. The first, alluded to 
above, refers to differences between artificial and natural modes of discrimination 
between wild-type and mutant cells. The second aspect concerns environmental 
effects on genome stability. Just as natural environments may reveal more complex 
and graded phenotypic changes than those that are commonly considered in the 
laboratory, nature may also act on genome stability in subtle ways, our experimental 
manipulations of this stability appearing crude in comparison. Current experimental 
models that appear to best mimic natural evolution are based on the study of 
bacteria in stationary phase. Indeed, stationary-phase populations are highly 
dynamic and generate extensive genetic diversity (Finkel and Kolter 1999). 

Approaches for the understanding of natural adaptation may also resort to 
comparative biology. One possible strategy would involve comparisons in the 
bacterial dimension. For example, differences may exist in the adaptive mode of an 
enteric bacterium, such as E. coli, and that of an isolate from Antarctic ice or a 
hydrothermal vent. A second, perhaps complementary, strategy, would be to evalu- 
ate adaptive responses along an environmental axis, graded, for example, 
from cold to hot, or from alkaline to acidic. In this manner, an understanding of 
bacteria as variable and responding components of their environments may become 
achievable. 

Along those lines, the present review tentatively proposes to consider extreme 
soils as applying a composite selection pressure, comprised of a complex and 



48 P. Dion 

heterogeneous soil component, and of a unique and defined extreme component. In 
this light, extreme soils may be perceived as combining the characters of both 
natural and experimental environments. The natural component would be represented 
by the soil matrix, with its complex environmental heterogeneity and spatial 
distance factors (Ramette and Tiedje 2007). For its part, the defining extreme 
parameter of extreme soils would introduce selection pressure akin to that produced 
in well-characterized experimental systems. Thus, an internal standard would 
become available, in the form of an adaptive response to a defining extreme 
parameter, to evaluate and compare evolutionary responses as they occur in 
different soils and in different organisms. 

The general forces shaping soil microbial communities are discussed in the next 
section. Then the particular case of extreme soils is presented, with an examination 
of the selective pressures at play and of the prokaryotic adaptive response to these 
pressures. Finally, these various elements are combined in the description of a 
prototypal fitness landscape that would be characteristic of extreme soils. 



3.4 Bacteria in Soils, a Substrate for Evolutionary Change 

Heterogeneity, functional complementarity, and intense communication are 
characters shared by both the soil and immune cell systems. These two systems 
have been constituted by a process of evolutionary layer accretion, corresponding, 
first, to the maintenance of environments along a geological or evolutionary scale, 
second, to the sustained coexistence and interaction of organisms or cells having 
appeared in succession in these various environments, and, third, to the perpetua- 
tion of mechanisms involved in symbiotic relationships (Dion 2008). 



3.4.1 The Soil Environment 

Soil heterogeneity is a multifactorial character (Giri et al. 2005). It results from the 
interplay of spatiotemporal, physical, chemical and nutritional variables delineating 
spheres of influence (Beare et al. 1995) that may separate bacteria with respect to 
location, physiology, or genetics. Heterogeneity contributes to diversification in 
populations (Kassen 2002), and this applies to the particular case of soils (Korona 
et al. 1994; Bardgett 2002; Torsvik et al. 2002). Conceivably, genotypic diversity 
might represent local adaptation to very specific niches, the continuous dispersal of 
locally nonadapted genotypes, or simply neutral variation (Vos and Velicer 2006). 
However, it seems well established that genetic patterns among soil microbial pop- 
ulations are influenced by habitat (Mc Arthur et al. 1988; Noguez et al. 2005), and 
particularly soil type (Gelsomino et al. 1999). 

The making of a soil bacterium involves both global genomic adaptations, such as 
adjustments in codon usage preference (Willenbrock et al. 2006), and fine tuning. 
An illustration of fine tuning is provided by the local adaptation of soil bacteria to 
trophic conditions found at a particular site (Belotte et al. 2003). Extensive adaptation 



3 Extreme Views on Prokaryote Evolution 49 

to soil conditions depends on expression of many properties, such as survival and 
stress responses, substrate utilization, chemotaxis, surface adhesion, interaction with 
plants and other microbes, and signalling pathways (Wipat and Harwood 1999). 

Adaptation and selection occur at very small scales in the soil heterogeneous 
substrate, as suggested by the observation of extensive diversity at the centimetric, 
millimetric, or even submillimetric scale (Grundmann and Normand 2000; Torsvik 
and 0vreas 2002; Vogel et al. 2003), coupled with the demonstrated capacity of 
particular clones to spread and become distributed in soils (Grundmann 2004). 
Microscale analysis indicates quantitative and qualitative heterogeneity within soil 
bacterial or archaeal communities (Nunan et al. 2002; Nicol et al. 2003). 

Soil colonization patterns are influenced by soil structure, as microbial 
communities established outside and inside aggregates show quantitative and 
structural differences. Most bacteria are located inside the aggregates or associated 
with the dispersible clay fraction (Ranjard et al. 2000). Clay and organic carbon- 
based colloids may facilitate microbial survival and growth by providing nutrients 
and protection against predation. Sand fractions appear to be preferentially colonized 
by fungi and by bacteria that resist grazing and that are adapted to nutrient limitations 
(Van Gestel et al. 1996; Ranjard et al. 2000; Sessitsch et al. 2001). 

In soil, bacteria are distributed in patches, the location of which may correspond 
to local substrate deposits stimulating growth and creating cell-density gradients 
(Nunan et al. 2003). This patchy distribution of growing cells may have a regulatory 
effect on bacterial activity, as a result of diffusion of substrates and metabolites 
(Grundmann et al. 2001). Metabolic interactions also contribute to structure soil 
microbial communities. For example, populations of nitrifying bacteria establish 
spatial associations of two bacterial types carrying out two successive biochemical 
transformations (Grundmann and Debouzie 2000). Bacteria collaborating in the 
stepwise oxidation of ammonia are located together in randomly distributed and 
preferentially colonized patches within the soil matrix; they are distributed 
nonrandomly within these preferentially colonized patches (Grundmann et al. 
2001). Hence, it is apparent that bacterial distribution is fine-tuned at the microscale, 
to reflect preferential colonization abilities and thus maximize adaptation. These 
small-scale spatial relationships are themselves influenced by various soil 
parameters, such as management practices (Webster et al. 2002). 



3.4.2 Bacterial Adaptations to Soil and the Role 
of the Stress Response 

Soil microbial selection is the result of a complex interplay of biotic interactions, 
involving competition for nutrients, antibiosis, and predation (Lockwood 1988; 
Hoitink and Boehm 1999), and of abiotic factors, the latter including lack of water 
or oxygen, nutrient starvation, exposure to toxic pH or metal cations, and high 
osmotic or matric tension (Van Veen et al. 1997). These selection components help 
establish the heterogeneous nature of the soil as a microbial habitat. They apply 



50 P. Dion 

external forces on the microbial community, shaping it while at the same time 
eliciting a biological response that is expressed as a capacity for self-organization 
(Young and Crawford 2004). Self -organization primarily arises as a byproduct of 
stress responses. Indeed, stress induced by environmental factors provides both a 
mechanism and a substrate for selection, because it both reduces fitness and elicits 
a controlled genetic instability leading to increased variability and the emergence of 
better adapted clones. In this manner, stress provides a dynamic and self-responding 
mechanism for shaping bacterial communities (Saint-Ruf and Matic 2006). 

A global stress response, resulting in the induction of more than 70 genes in 
reaction to a variety of physical and chemical stresses (Hengge-Aronis 2002), is under 
the control of a specialized sigma factor, such as RpoS in E. coli (Hengge-Aronis 
1999) or SigB in Bacillus subtilis (Price et al. 2001). RpoS interferes with substrate 
utilization, leading cells to balance their capacity for substrate utilization and stress 
resistance through mutation of the rpoS gene (King et al. 2006). High intracellular 
concentrations of RpoS increase mutation rates by downregulating the mismatch 
repair system and the dinB gene, coding for the Pol IV translesion synthesis DNA 
polymerase (Saint-Ruf and Matic 2006). In addition to the general stress response, 
E. coli also exhibits a mutagenic SOS response in reaction to extensive DNA dam- 
age and inhibition of DNA synthesis (Janion 2001; Michel 2005). This response, 
under the control of the LexA repressor and the RecA coprotease, involves the pro- 
duction of error-prone DNA polymerases II, IV, and V responsible for translesion 
synthesis (Napolitano et al. 2000). It has been suggested that a variety of environ- 
mental stimuli, such as starvation, could elicit the SOS response by endogenous 
induction of DNA damage (Aertsen and Michiels 2006). Antibiotic-induced defec- 
tive cell wall synthesis has also been identified as an inducer of the SOS response 
in E. coli (Miller et al. 2004). 

Microbial populations may reconfigure themselves through mutation and natural 
selection, so as to reduce stress. Mutations improve the correspondence between 
environmental factors and physiological requirements, through adjustment of either 
the mutating cell itself or else its surroundings, the latter effect resulting from 
bacteria-induced microenvironmental changes (Banas et al. 2007). In a second- 
order response, the ability to reduce stress through mutation is itself a selectable 
trait (Earl and Deem 2004), with a general evolutionary trend towards a reduction 
of the standard mutation rate, and allowing higher mutation rates under specific 
circumstances (Drake et al. 1998). This results in the acquisition of stress-induced 
and transiently expressed "evolution- accelerating systems" (Kivisaar 2003). 



3.4.3 The Stress Response as a Mechanism 
and an Object of Evolutionary Change 

The existence of an evolved genetic instability response to environmental stress is 
suggested by three sets of observations. The first concerns apparently divergent 
responses of an organism to stress, with stress-induced mechanisms for both increased 



3 Extreme Views on Prokaryote Evolution 5 1 

or decreased genetic instability having been identified in E. coli and a variety of 
environmental bacteria (see below, this section). A second observation is that 
orthologous stress-related proteins have divergent effects on gene stability in different 
organisms. The most notable example is RecA, whose action promotes hypermutabil- 
ity in E. coli, but prevents it in Thermus thermophilus (Castan et al. 2003). Perhaps 
in keeping with these differences in function, recA is diversely regulated in E. coli 
and other organisms (Mazon et al. 2006). A third observation suggesting evolution of 
genetic instability in bacteria is that differences exist in intrinsic genetic stability in 
different organisms. For example, it has been suggested that the hyperthermophile 
crenarchaeon Pyrobaculum aerophilum is deficient in mismatch repair and has devel- 
oped the ability to survive as a permanent mutator (Fitz-Gibbon et al. 2002). By con- 
trast, spontaneous mutations occur at low rates in the thermoacidophilic archaeon 
Sulfolobulus acidoalcarius (Grogan et al. 2001). Thus, it appears that the mutagenic 
response to stress is subject to second-order selection (Bjedov et al. 2003) and intense 
diversifying evolution (Saint-Ruf and Matic 2006). 

As a result of this second-order evolutionary process, bacteria have acquired the 
capacity to switch phenotypically between high and low mutation rates depending 
on environmental conditions. Several such switches have been postulated to exist 
in bacteria, and the functioning of some of these may involve the RpoS-mediated 
general stress response (Bjedov et al. 2003). They allow acquisition of a high 
mutation rate, followed by a return to genetic stability. In addition to occurrences 
where they hitchhike beneficial traits to fixation in small asexual populations 
(Sniegowski et al. 2000), mutator phenotypes will be selected when gene diversity 
limits adaptation. This may occur, for example, following the introduction of a 
strong stress-generating parameter. However, mutator phenotypes are counterse- 
lected once adaptation is achieved, as a result of continuous production of 
deleterious mutations (Denamur and Matic 2006). On broad terms, phenotypic 
switching and return to low mutation rates may be rare or intense. Rare switching 
occurs when the mutator phenotype is stably expressed as a result of a mutation in 
an antimutator gene, and in this case reduction of the mutation rate may be achieved 
by reversion, secondary mutation, or reacquisition of the wild-type antimutator 
allele by horizontal gene transfer (Denamur and Matic 2006). 

Intense phenotypic switching occurs when the mutator phenotype is transiently 
expressed through physiological adjustments. Indeed, a variety of physiological 
and genetic mechanisms contribute to modulate the level of genetic stability as a 
function of environmental factors (Table 3.1). 

An examination of the structure of soil microbial communities (see Section 
3.4.1) suggests that, in soils, stresses are spatially and temporally heterogeneous, 
and that, correspondingly, the bacterial stress response is patchy and localized. 
Hence, soil microbial communities can be viewed as an assemblage of individuals 
responding to various levels of stress by modulating genetic stability. Genetic insta- 
bility becomes advantageous when maladaptation increases the potential benefit of 
mutation. In turn, expression of a newly produced beneficial mutation will result in 
alleviation of stress, which can then be viewed as a physiological relay between the 
environment and cell determinants of genetic stability. 



52 



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56 P. Dion 

Within the highly structured and heterogeneous soil environment, a defining 
extreme parameter would act as a cross-gradient factor. Thus, application of this 
extreme parameter will attenuate the heterogeneous character of the soil environ- 
ment, as a result of cross-protection (Jenkins et al. 1988; Hengge-Aronis 2002) that 
is provided by response to one particular stress (such as that caused by the defining 
extreme parameter) against unrelated stresses (such as soil-associated stresses). It 
may also be envisioned that there exists a maximal level of induction of the stress 
responses through the RpoS and RecA/LexA regulation, and that superimposition 
of a defining extreme parameter will result in constant induction of these responses 
at maximal or near-maximal levels. In this manner, the heterogeneous character of 
the soil, which was originally expressed as variations in stress levels according to 
space and time, will be tamed. 



3.5 The Context of Extreme Soils 

Initial land colonists might have been confronted with harsh conditions, characterized 
by intense radiation and low moisture. Whether early soil colonization was effected 
by thermophiles is debatable. It has been proposed that, along the history of the 
Earth, biotic evolution was tightly coupled with that of climate (Schwartzman and 
Lineweaver 2005). Climate might have been as warm or warmer in the Archean 
than today, and possibly very hot (Des Marais 1998; Kharecha et al. 2005). Surface 
Archean temperatures were estimated to have been ~55-80°C (Knauth and Lowe 
2003), and the oceans might have been at a temperature of 70°C in the Precambrian, 
for much of the time between 3.5 and 2.5 billion years ago (Ga; Knauth 2005; 
Robert and Chaussidon 2006). Hence, it may be that initial land colonizers were 
thermophiles. At later times, major glaciation events, occurring at about 2.4 Ga and 
between 0.8 and 0.6 Ga (Kirschvink et al. 2000), may have resulted in massive 
psychrophilic adaptations (see Chapter 2). 

In addition to global changes, local perturbations, such as the initiation of 
geothermal or volcanic activity (see Chapter 8) or else a change in the course of an 
acidic or heavy metal-rich stream, may superimpose an extreme character on a soil. 
The advent of a defining extreme parameter can have an abrasive or thinning effect 
on soil evolutionary layers, decreasing the number of species or individuals 
represented within a particular evolutionary layer (see Chapter 4), or removing 
some layers altogether when a particular metabolism becomes impossible. This 
would be the case, for example, of obligate photoautotrophy, as photo synthetic 
activity is inhibited above 73°C (Brock 1985; Rothschild and Mancinelli 2001). At 
the same time, this extreme parameter will have a founding effect, as the process 
of evolutionary layer accretion will be reinitiated on the basis of novel mutual 
dependences. 

In extreme soils, a single defining parameter is superimposing a strong selective 
pressure on the soil system. The nature of this defining extreme component varies, and 
correspondingly the physiological response to this component also varies. The reader 



3 Extreme Views on Prokaryote Evolution 57 

is referred to other chapters in this volume (see Chapters 2, 5, 8, 10, and 16) and 
various reviews, some examples of which are given below, for descriptions of 
physiological adaptations to heat (Stetter 2001), cold (D' Amico et al. 2002; Scherer 
and Neuhaus 2006), radiation (Battista et al. 1999; Baumstark-Khan and Facius 
2001), extreme pH (Baker- Austin and Dopson 2007), and salt (Kunte et al. 2001; 
Oren 2006). 

Given the complexity and sophisticated character of the adaptive responses to 
various extreme conditions (Cleaves and Chalmers 2004), it may be debatable 
whether such adaptations appear de novo in a soil newly exposed to extreme 
conditions. Certainly, extremophiles may pre-exist in soils in advance of the 
superimposition of extreme conditions (Norris et al. 2002), and some micro-organisms 
can be transported over considerable distances (Griffin et al. 2001; Kellogg and 
Griffin 2006). However, there are numerous examples to show that dispersal does 
not impede microbial diversification and the appearance of endemic populations 
(Whitaker and Banfield 2005). More specifically, it has been proposed that the bio- 
geographic distribution of micro-organisms results from a balance between origina- 
tion and extinction processes, with the environment being in part responsible for the 
spatial variation in microbial diversity (Martiny et al. 2006). 

A study of the diversity and biogeographic distribution of soil bacterial 
communities across the American continent led to the conclusion that distribution 
is influenced primarily by edaphic variables, with soil pH playing a major role in 
the nonextreme soils that were examined (Fierer and Jackson 2006). Thus, the 
changes that are considered here do not correspond exclusively to the genetic 
creation of heterogeneity. The response to a recently introduced soil extreme 
component would be defined as a balance between selection of pre-existing 
colonists and generation of newly adapted forms. In discussing the respective 
contributions of dispersal and adaptation in establishment of biogeographic diver- 
sity patterns, it must be remembered that combinatorial processes occurring in 
mosaic genome pools enhance the adaptive potential of populations beyond that of 
individuals that compose them (Allen et al. 2007). 

Although physiological adaptations to the various environmental extremes 
appear to be vastly different, commonalities become apparent when basic 
mechanisms and initial responses are considered. At the physiological level, 
changes in plasma membrane potential and/or ion flux modifications are amongst 
the earliest cellular events in response to a great variety of stresses and biological 
or mechanical stimulations (Shabala et al. 2006). At the genetic level, the stress 
response is a shared trait, universally expressed in prokaryotes and eukaryotes. 
Archaea share some components of the stress response with bacteria and others 
with eukaryotes (Macario et al. 1999). The universal stress response involves a core 
of common genes, and a minimal stress response proteome comprising 44 proteins, 
including the bacterial RecA or its eukaryotic homolog Rad5 1 , has been postulated 
to exist in all cellular organisms (Kiiltz 2005). Of course, other components that 
complement this minimal protein set vary between organisms, and the modes of 
action of common components also vary. For example, the activity of RecA 
orthologs differ, as mentioned earlier (see Section 3.4.3). 



58 P. Dion 

The generalizations that can be drawn at the level of the basic microbial 
responses suggest that commonalities also exist between evolutionary processes. In 
particular, adaptation to edaphic and extreme environmental factors may involve 
similar mechanisms and influence each other, exerting a combined influence on 
resident bacteria. This compound influence is also noticeable at the level of 
community structure, as both the soil (Cho and Tiedje 2000) and extreme 
components (Whi taker and Banfield 2005) of the environment contribute to 
isolation of the resident populations. Indeed, isolation favors the creation of island 
dynamics, introducing into populations geographically restricted prokaryotes, or 
geovars (Staley and Gosink 1999), carrying environment- specific genes (Tringe 
et al. 2005). 

The combined action on stress-responsive mechanisms of the adapting cell 
remains decomposable into its two constitutive elements. This implies that, 
although influencing the same target processes, the soil and defining extreme 
parameters may act synergistically or antagonistically on adaptative change (see 
also the discussion on epistasis below, Section 3.6.1). 

Evolution in extreme soils can be envisioned to occur in two possible directions, 
which are simply described as "soil to extremophiles" and "extremophiles to soil". 
In the first case, a soil and its resident microbial community become exposed to a 
defining extreme parameter. In the second case, extremophiles from an extreme 
nonsoil environment colonize a soil sharing the extreme property. Hence, evolution 
of soil extremophiles occurs in a two-dimensional vectorial space, organized along 
the soil component and the extreme component axes. Assuming that an organism 
is already optimally adapted to soil, then evolution will occur strictly towards extre- 
mophily. Reciprocally, an optimally adapted extremophile will solely evolve soil 
microbial characteristics. In practice, however, departure from optimality is 
expected to occur in the course of adaptation (Fig. 3.1), as the result of genetic and 
physiological constraints as discussed below. 



3.6 The Extreme Soil Adaptive Landscapes 
3.6.1 Description of the Landscape 

The relationships between environment and genotype have been described in terms 
of adaptive landscape (Wright 1988). This landscape can be viewed as a series of 
peaks occupied by optimally fit genotypes, separated by valleys where genotypes 
are not well adapted to their environment. Epistasis, or the interaction among 
genetic loci in their effect on phenotypes, results in linkage disequilibria (Maynard 
Smith et al. 1993) and influences the migration of populations in the adaptive land- 
scape (Whitlock et al. 1995). Thus, as adaptation proceeds and nonrandom associ- 
ation of alleles and unlinked loci occurs, individuals become increasingly 
committed along evolutionary paths leading to a particular fitness peak. This can be 



3 Extreme Views on Prokaryote Evolution 



59 



Fig. 3.1 Simplified view of patterns of evo- 
lution for soil micro-organisms and extremo- 
philes colonizing extreme soils. The horizontal 
shaded arrow indicates that organisms already 
adapted to soil conditions evolve along a 
fitness gradient leading to tolerance of a 
defining extreme soil parameter. The vertical 
shaded arrow suggests that, reciprocally, 
organisms already adapted to extreme condi- 
tions acquire soil- specific adaptations. The 
dashed arrows illustrate that both soil organ- 
isms and extremophiles might lose some of 
their original adaptations in the course of 
this process, through tradeoffs or other 
mechanisms 



o 

c 
o 



Q 
CO 



Adaptation of soil organisms 




Selection for extreme character 



envisioned as a nth-order Markovian process (Usher 1979), whereby a transition 
from one genotype to another is influenced by an increasing set of previous geno- 
types, and hence becomes increasingly constrained (Fig. 3.2). 

Peaks of adaptive landscapes can be sharp or smooth, depending on whether 
they are defined by strong or weak selection pressure, and they can be occupied 
simultaneously by a few or several different genotypes. Rugged fitness landscapes 
may present an initial nonadapted population with many possible peaks to climb, 
and under these conditions chance events are important in determining the initial 
evolutionary course (Korona et al. 1994; Colegrave and Buckling 2005). 

The nature of evolutionary events differs in sharp and smooth peaks (Peliti 
1997). In the first case, there are strong epistatic interactions, as the strength of 
directional epistasis is correlated with the average effect of a single mutation 
(Wilke and Adami 2001). This correlation implies that mutations with strong fit- 
ness effects will synergistically or antagonistically affect each other, hence defining 
the two signs of directional epistasis. On the other hand, in smooth peaks epistatic 
interactions are not so prevalent initially, but there is strong influence from Muller's 
ratchet, as slightly deleterious mutations tend to be fixed during evolution in asex- 
ual populations (Peliti 1997). The robustness of a system, or its tolerance to delete- 
rious mutations, is correlated with antagonistic epistasis. Furthermore, robustness 
defines a threshold beyond which synergistic epistasis occurs, where the combined 
effect of co-existing mutations is larger than expected from the addition of their 
individual effects (Bershtein et al. 2006). Hence, whether they are expressed imme- 
diately or only after a robustness threshold is reached, synergistic epistatic interac- 
tions will multiply the effect of deleterious mutations, in both a smooth and a 
rugged fitness landscape. 

Heterogeneity in soils provides for rugged fitness landscapes, with many sharp 
peaks. Sharpness of the peaks results from a variety of factors determining requi- 
sites for soil adaptation. Upon imposition of a defining extreme condition, the soil 
component of a fitness peak is expected to flatten, as the correspondence between 
heterogeneous edaphic factors and the intensity of the stress response loosens (see 



60 



P. Dion 



Initial 

absorbing 

condition 










\Horizontal transfer 










Final 






absorbing 


5*. ^r"" - **. -77— £l . z^z * 




conditions 

x, 








^ x 4^.,^..,^ 







Markovian 
transition 



Subsequent events of 
committed evolution 



Selection pressure : 



Decreased gene 
damage 



Fig. 3.2 Diversification of an initial population, occurring within a Markovian dependency 
structure of evolutionary changes, and under selection pressure for decreased gene damage. 
Evolutionary changes establish a nth-order Markovian chain, in which a given evolutionary con- 
dition X r (r = 1, 2, 3, ..., n) at a given time depends on an elongating set of previous evolutionary 
conditions established at sets of previous times, up to the initial absorbing condition X q . In this 
way, the stochastic evolutionary process becomes increasingly constrained and committed, 
through epistatic interactions and the consequent generation of linkage disequilibrium. Upon 
completion of the adaptation process, each of the final absorbing conditions X corresponds to the 
summit of a particular fitness peak. In the figure, the increased commitment of evolution is indi- 
cated as a wavy line of Markovian transition, which in reality would occur gradually. Horizontal 
transfer between evolving individuals would promote shifting from one Markovian evolutionary 
path to another 



Section 3.5 for a discussion of the combined effects of edaphic and extreme 
parameters in influencing the stress response). Other factors contributing to the 
flattening of soil fitness peaks may include an expected decrease in intensity of 
biological interactions, as fewer soil organisms would tolerate the newly estab- 
lished defining extreme condition. More generally, there will be an equilibration of 
genotype fitness and adaptation capacity, as initially well-adapted genotypes might 
have become less capable of further adaptation to the newly changed extreme envi- 
ronment. Indeed, it has been observed that niche specialization may come with a 
cost of reduced potential to diversify (Buckling et al. 2003). Antagonistic pleiot- 
ropy, arising from tradeoffs (Cooper and Lenski 2000), might also contribute to 
make some of the better soil colonists less capable of adapting to the defining 
extreme condition. 

The importance of biological interactions in defining sharpness of fitness peaks 
in the soil adaptive landscape is evidenced by the difference in capacity for survival 



3 Extreme Views on Prokaryote Evolution 61 

of human intestinal pathogens in autoclaved and nonautoclaved soils (Jamieson 
et al. 2002; Jiang et al. 2002). It can be expected that the diminished microbial 
populations and partial or complete removal of plant hosts or other partners would 
contribute to flatten the fitness landscape by abolishing opportunities for competition, 
predation, and mutualism. 



3. 6.2 Lessons from Toxified Soils 

The results of studies on herbicide application to soil illustrate the effect of stressors 
on soil microbial communities. In one such study, herbicide application resulted in 
a shift in microbial community composition, as some microbial species became less 
prevalent whereas others were stimulated (Engelen et al. 1998). This may be 
interpreted as a decrease in the impact of the soil component on community 
structure, as some genotypes that normally would be away from fitness peaks as 
defined by the soil factors had an opportunity to become dominant. These particular 
genotypes were less subjected to soil constraints and responded to a novel selection 
pressure imposed by the herbicide. Similarly, treatment of soil with Zn resulted in 
an initial death of microbes due to metal toxicity, followed by regrowth of metal- 
tolerant bacteria (Diaz-Ravina and Baath 1996). There are numerous other instances 
where some groups of soil micro-organisms increased in numbers or proportion 
following chemical disturbance with heavy metals, pollutants, or herbicides (Kent 
and Triplett 2002). 

If indeed the enhanced growth of some microbial groups in toxified soils reflects 
a decrease of the relative importance of the soil component of selection and a 
flattening of the corresponding fitness peaks, then there should be instances where 
a particular toxic treatment would result in overall decrease of microbial biomass, 
but without affecting microbial diversity. This was in fact observed in some 
instances. For example, treatment of soil with the fungicide triadimefon caused a 
decline in organic carbon and soil microbial biomass but no decline in microbial 
DNA diversity as measured with RAPD random primer amplification (Yang et al. 
2000). In this case, the fungicide treatment may be viewed as having decreased the 
fitness of dominant bacteria residing at the summit of fitness peaks, thus causing an 
overall decline in microbial biomass, while allowing a greater number of previously 
maladapted genotypes to co-occupy the summit of smoother fitness peaks. 
Similarly, soil microbial biomass was more sensitive to pollution with heavy metals 
than was microbial diversity (Kandeler et al. 2000), again suggesting a relief from 
edaphic determinisms upon imposition of a defining extreme parameter. However, 
the above-cited studies remain specific in scope, and it is agreed with Kent and 
Triplett (2002) that more should be known on the relationship between amount of 
biomass and diversity of soil microbial communities. 

Thus, introduction of a defining extreme condition in soil results in a remode- 
ling of the adaptive landscape. This landscape now comprises composite fitness 
peaks, with a smooth soil component and a sharp component defined by the 



62 



P. Dion 



extreme parameter (Fig. 3.3). Initially, a variety of different soil bacteria will 
reside on the smooth soil component peak. Being away from the ultimate summit 
defined by the extreme condition, soil bacteria will be subjected to stress and 
enhanced mutagenesis. In this sense, they will go through a dual selection proc- 
ess, as they are presented with both the edaphic and the extreme components of 
fitness peaks. In both cases, selection for decreased stress and enhanced genetic 
stability will be exerted, whereas other organismic processes, such as growth and 
biotic interactions, will not be as strictly monitored as they are in nonextreme 
environments. 

The extreme component exerts a homogenizing effect on the soil environment 
(see Section 3.6.1). In this sense, fewer fitness peaks will subsist in extreme soils 
(Fig. 3.4), bringing various genotypes into coexistence and providing for novel 
interactions. Numerous examples of interactions occurring on the fitness peaks 
defined by the extreme parameter are to be found in the present book. In particular, 
the reader is referred to descriptions of food webs created in extreme soils (see 
Chapter 4), of lipolithic and endolithic communities from arid soils (Wierzchos et 
al. 2006; see Chapter 6), of geochemical cycling in cold soils (see Chapter 7) and 
peatlands (see Chapter 9), and of cometabolic relationships established in hydrocar- 
bon-contaminated desert soils (see Chapter 13). 



B 



Fully adapted population 

L 




II. Sharp peak evolution 
I. Smooth peak evolution 




Extreme component 



\^__ Soil component 

1 



Fig. 3.3 Shape of an individual fitness peak from the adaptive landscape of a nonextreme soil (A) 
and an extreme soil (B). In nonextreme soils, fitness is determined by a complex series of edaphic 
and environmental, including climatic, parameters. These interact to mold a fitness landscape 
composed of simple peaks such as the one shown in (A). According to this simplified fitness 
topology, a microbial population optimally adapted to one of these peaks would have to lose some 
of this adaptation if it were to move to another peak and adapt to a different niche. In extreme 
soils, decreased intensity of interactions and other factors reduce the sharpness of the soil compo- 
nent of composite fitness peaks. Thus, different populations can coexist in a particular soil niche, 
but also have to adapt to a defining extreme parameter. In extreme soils, adapting populations 
must follow a path indicated by the arrows, to the summit of composite fitness peaks comprising 
a flat soil component and a sharp extreme component 



3 Extreme Views on Prokaryote Evolution 



63 



Homogenizing extreme 
component 



f 



Population adapting to 
extreme component 



Heterogenous 
soil component 




Population adapting to soil 
component 



Fig. 3.4 Composite topography of adaptive landscape of an extreme soil. The extreme compo- 
nent of composite fitness peaks acts as a unifying factor, through the creation of new and restricted 
environments where resident populations are brought together and compete. On the other hand, 
the extreme component also acts as a discriminating factor, whereby some particular soil adapta- 
tions may be incompatible with further adaptation to the defining extreme parameter. This creates 
evolutionary dead-ends, in the form of soil fitness peaks which are not connected to the superim- 
posing extreme fitness peak 



3.7 Conclusions 

The evolutionary effect of mutations varies according to the position of a genotype 
in the fitness landscape. To bacteria finding themselves at the bottom of a peak, 
mutation offers the promise of adaptation, and environmental challenges create 
pressure for greater evolvability of individuals. As the genotypes climb the fitness 
peak, selection for genetic stability occurs. Thus, it appears that evolution acts on 
evolution itself, in a mirroring effect. In extreme soils, where the selection pressure 
is in part defined by a clearly identifiable parameter, the mirror of evolution might 
provide illuminating images. There is truth to be found in such soils, in the absence 
of those distractions that gentler environments provide. 

Acknowledgements The author is thankful to Ziv Arbeli for critical reading of the manuscript. 



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Chapter 4 

Biodiversity: Extracting Lessons 

from Extreme Soils 



Diana H. Wall 



4.1 Introduction 

The organisms that live in extreme environments have justifiably captured the 
imagination of people fascinated with the detection of life and exploration. Reasons 
for this captivation vary. Some see exploration of these organisms and their envi- 
ronment as a scientific area to provide insight about life on earth, whereas others 
see economic potential. Whether the extreme environment is human-caused, such 
as a polluted soil, or a more natural environment (aquatic hot springs, ice, ocean 
depths, atmosphere, or land), unravelling and understanding the resident organ- 
isms, their mechanisms of survival, and the intricate relationship between the habi- 
tat and other species, can help us understand life on this planet and elsewhere. 
Because of global changes, many aspects of extreme environments, such as the 
identity and types of organisms and communities, the biological traits that allow 
evolutionary success in a harsh environment, the patterns of distribution of these 
organisms, the factors controlling their distribution, and their influence on and 
feedback from ecosystem processes, have increasing relevance to all terrestrial 
ecosystems. This chapter examines how extreme soils as a habitat for biota can 
inform our general knowledge of terrestrial biodiversity in many other ecosystems. 
A brief background on soil biodiversity from other terrestrial systems is presented 
to set the stage for lessons derived from studies of extreme soils. 

Biodiversity is defined by the United Nations Convention on Biodiversity 
(CBD) as the "variability among living organisms from all sources . . . and the bio- 
logical complexes of which they are a part: this includes diversity within species, 
between species and of ecosystems" (Convention on Biodiversity 2004). This 
expansive definition is extremely useful for describing life on earth, determining 
the biotic composition of an ecosystem, and addressing the rapid changes occurring 
at temporal and spatial scales to the ecosystem, such as the increasing rate of 



Diana H. Wall 

Department of Biology and Natural Resource Ecology Laboratory, Colorado State University, 

Fort Collins, CO 80523-1499 

e-mail: Diana@nrel.colostate.edu 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 71 

© Springer- Verlag Berlin Heidelberg 2008 



72 D.H. Wall 

extinctions of species. Scientists have emphasized that entire populations, as well 
as single individuals of a species, are being lost at an accelerating rate. The CBD 
definition is based on both classical morphological and/or genetic taxonomic 
knowledge of the biological distribution of species, whether endemic or widespread. 

The numbers of species of plants and large animals across landscapes and their 
global distribution are better known than for smaller invertebrates or microbes, and 
likewise, more is known about biodiversity above- than belowground (Wardle 
2002). Whether there are accelerating rates of extinction for less visible organisms 
such as bacteria or fungi, and particularly for belowground biota, has yet to be 
determined. Instead, in many cases, protection of a land area for aboveground 
species assumes belowground species are also conserved. Whether the spatial scale 
of the protected area is adequate for conservation of both above- and belowground 
species and food webs is less studied, but land conserved for a plant in a small habi- 
tat might be inadequate to conserve significant levels of diversity belowground. 

The world's soils have a large abundance and wealth of biotic diversity with 
species numbers estimated to be greater than aboveground diversity (Wardle 2002). 
Taxa include microbes (bacteria among which are cyanobacteria and actinomyc- 
etes, fungi, Archaea), protozoa, microscopic invertebrates (microarthropods, nema- 
todes, rotifers, tardigrades), large invertebrates (snails, millipedes, centipedes, termites 
and earthworms), vertebrates (moles, gophers, lizards), vascular plant roots and 
lichens, cryptogamic crusts, algae, and mosses. Many of these smaller groups can 
be found in a handful of soil (Wall and Virginia 2000). Because of the abundance 
and diversity of the multiple taxonomic groups, identifying all species and their 
interactions in a single soil sample has been problematic. Instead, our understanding 
of soil biodiversity is largely based on trophic or functional classifications (e.g., 
herbivore, predator, microbial feeder, detritus feeder) derived from scientific litera- 
ture on the feeding habits and morphology of a few species. This approach can then 
be extrapolated to assemble other unnamed species into functional groups in com- 
plex food webs. The contribution of soil biota components in ecosystem processes 
has thus been postulated based on functional grouping of similar species into food 
webs. This has proven useful to quantify the role of soil biota in processes such as 
regulating the rate of soil organic matter decomposition, nitrification, primary pro- 
duction, and nutrient cycling (Hunt and Wall 2002). However, research is needed, 
as assumptions regarding functionality of large groups of soil organisms may not 
provide realistic measures of these processes. 

A further and critical recognition of the dependence of humans on the benefits 
provided by soil biodiversity is the concept of ecosystem services (Millennium 
Ecosystem Assessment 2005; Wall 2004). These include carbon sequestration, 
generation and renewal of soil structure and soil fertility, flood and erosion control, 
bioremediation of wastes and pollutants, modification of the hydrologic cycle, 
regulation of atmospheric trace gases, and biocontrol of human, animal, and plant 
pathogens and parasites. Alterations and loss of the world's terrestrial soils are 
occurring rapidly, raising concerns that some of these services may be largely 
interrupted, as they are currently rendered by unsustainable soils (Millennium 
Ecosystem Assessment 2005). 



4 Biodiversity: Extracting Lessons from Extreme Soils 73 

Given this background on soil biodiversity, can soil biodiversity of extreme 
environments inform us about biodiversity and ecosystems elsewhere? My intent 
here is to augment lessons from microbes living in extreme soils with examples of 
their consumers, primarily invertebrates, in order to better extract and extend 
lessons to biodiversity inhabiting global soils. It is hoped that these lessons will be 
further expanded and clarified by the many scientists who are developing exciting 
new approaches for detecting these amazing organisms and learning how they live 
in extreme soils and are integral to the working of ecosystems. 



4.2 Lesson One: Biodiversity in Soils Is Hidden 

Although this statement appears obvious to those working on extreme soils, scien- 
tists often bring their biases of larger, visible, and more easily detected organisms 
to the study of soils. However, most life in soils is microscopic. In extreme soils, 
an emphasis is to detect and study microbes and microscopic life, particularly at the 
species or molecular level, whereas in other ecosystems the attention to larger, 
mostly visible life sometimes dismisses the variety of life below the surface. 

In extreme soils of lower plant and animal species diversity, multiple techniques 
are used to detect life. Without familiarity and testing of the correct extraction 
technique, microscopic animals and microbes may be considered absent and soils, 
'sterile'. Scientists working on extreme soils recognize that techniques used to 
isolate microscopic invertebrates are varied and require a basic understanding of 
the limitations of the method as well as the general biology of each particular 
group. Techniques for extraction, identification, and enumeration (based on classical 
morphology) are specialized and may differ for each group of taxa occurring in 
soils. For example, to extract microarthropods and nematode roundworms, two groups 
of mesofauna that occur in soils worldwide, a single technique should not be used. 

Microarthropods (mites and Collembola) live in air-filled pores of soil whereas 
nematodes are aquatic animals living in water films around soil particles. 
Nematologists may extract nematodes from soil with methods depending on soil 
type and organic matter content and whether they want to recover the whole com- 
munity or just a targeted species. Such methods are based on movement in water 
by gravity, sieving-centrifugation, or flotation techniques. Microarthropods have a 
different physiology and behavior and are removed from air pores in soil by methods 
based on active avoidance (e.g., avoidance of heat using Berlese-Tullgren funnels), 
aspiration, and flotation (Coleman et al. 1999; Ducarme et al. 1998). Within these 
two major groups of soil fauna, species differ in body size, movement, life histories, 
temperature requirements, feeding habits, and physiologies. In addition, many species 
are rare, and may not be detected without prior evaluation and use of several tech- 
niques. For example, the drier, saltier, low carbon soils of the Antarctic Dry Valleys 
(see Chapters 2 and 12) located away from meltstreams were considered almost 
sterile until the early 1990s, but different extraction techniques for nematodes and 
molecular analyses of microbes have shown greater diversity and distribution than 



74 D.H. Wall 

previously thought (Aislabie et al. 2006; Barrett et al. 2006; Freckman and Virginia 
1997; Wall and Virginia 1999). Rapid faunal analysis from soil using bulk animal 
DNA for identification is emerging as an addition to classical morphological tech- 
niques. However, as with microbial molecular tools, faunal DNA analysis will 
need to be related to activity of viable populations. As with extreme soils, using 
numerous techniques in a coordinated manner will increase detection of organisms in 
all terrestrial soil systems, no matter the location or depth of the soil. This informa- 
tion will increase our knowledge of Earth's biodiversity. 



4.3 Lesson Two: Soil Species Have More Than One 
Survival Strategy 

Many survival strategies exist among the organisms in extreme soils that may contrib- 
ute to evolutionary success. Distantly related organisms may share a strategy, and 
additionally may have developed multiple adaptations for maintaining populations. 
Evolution has selected for biota that express ecological traits such as long versus short 
life cycles, sexual versus other reproductive modes, numerous versus few eggs, mul- 
tiple dispersal mechanisms, alterations in morphology, and active migration to avoid 
stress. Types of cryptobiosis, an ametabolic, reversible response to environmental 
stress known in many taxonomically distinct organisms such as most tardigrades, 
rotifers, and nematodes, are a response to desiccation (anhydrobiosis), freezing (cryo- 
biosis), and salinity (osmobiosis; Block 1982; Pugh and Dartnall 1994; Sinclair and 
Sjursen 2001; Treonis and Wall 2005). In the Antarctic, soil nematodes have a variety 
of strategies including anhydrobiosis, cryobiosis, cold-hardiness (Pickup 1990), intra- 
cellular freezing (Wharton 2003), dispersal by wind (Nkem et al. 2006), and life histories. 
Microarthropods can supercool (Convey et al. 2003), be heat tolerant, or cold-hardy 
(Sinclair and Sjursen 2001), and can desiccate (Montiel et al. 1998; Worland and 
Lukesova 2000). Algae and mosses in extreme hot and cold deserts desiccate without 
water and in the polar deserts become freeze-dried through the long winters until 
temperature and moisture combine to trigger activity (McKnight et al. 1999). Examples 
of resistance mechanisms for microbes living in extreme soils are discussed throughout 
this volume, and add to the synthesis of the underlying evolutionary adaptations of 
all soil biota. 

Survival mechanisms extend to more biodiverse soils in other ecosystems. Even 
within a diverse phylum such as nematodes, anhydrobiosis is widely distributed. 
Many nematodes in temperate and tropical soils undergo anhydrobiosis when soils 
dry, including phylogenetically different species such as the fungal-feeding nema- 
tode Aphelenchus avenae (Browne et al. 2004; Crowe and Madin 1975; Freckman 
et al. 1980), bacterial-feeding species, Panagrolaimus and Acrobeloides, the obli- 
gate plant parasites Rotylenchulus reniformis and Scuttellonema brachyurum, and 
many others (Demeure et al. 1979; Goyal et al. 2003). However, the degree to 
which anhydrobiosis, like other forms of cryptobiosis, protects different species 
can vary (Rothschild and Mancinelli 2001; Wharton 2003). 



4 Biodiversity: Extracting Lessons from Extreme Soils 75 

Thus, a combination of ecological and physiological traits has allowed species 
to successfully evolve and maintain active populations in extreme soil habitats. 
These few examples from extreme soils suggest multiple strategies that might also 
be expressed in nonextreme soils to enable responses to environmental change. 



4.4 Lesson Three: Extreme Soils Are Ecosystems 

Although there may be fewer species in extreme soils, these particular soils harbor 
all the characteristics of an ecosystem, for example, species variability, food webs, 
nutrient cycling, production, decomposition, and interaction with the environment. 
Food webs in extreme soils are simpler than in other ecosystems and usually have 
fewer trophic levels because of lower energy or primary production input. In 
extreme soils, controls on trophic levels in a food web are dependent more on abiotic 
controls than on top predators of lower trophic levels. Whether the food web is 
primary producer-based or detritus-based, most food webs will involve only two 
functional groups, microbes and their consumers (Moore and de Ruiter 2000). 
Detrital-based food webs could have an additional trophic level if they depend on 
two types of contemporary primary production: autochthonous (algae growing in 
soils) or allochthonous (detritus blown in from a nearby source); but if based on 
ancient legacy carbon alone, there will be only two trophic levels. Protozoa and 
larger-sized organisms (of size range from 500 |im to 2mm), such as microfauna 
(rotifers, tardigrades) and mesofauna (microarthropods, nematodes) consume 
producers (cyanobacteria or algae), or consume decomposers (bacteria or fungi), 
and thus regulate the turnover of microbes and nutrients. As these organisms die, 
organic carbon and nutrients are recycled back to the soil. In some extreme soils 
such as in the Atacama Desert with their hypolithic communities of phototrophs, 
the organisms appear to interact solely as primary producers, but more data on 
heterotrophic microbes are needed (Warren-Rhodes et al. 2006; see also Chapter 6). 
The Antarctic Dry Valleys provide examples of soil communities with few spe- 
cies in trophic groups. These have both primary producer-based and detritus-based 
food webs. These include algal feeders - a single species of nematode, Eudorylaimus 
antarcticus (Wall 2007), bacterial feeders - two nematode species, and more rarely, 
fungal feeders - a mite and a collembolan species. Tardigrades and rotifers that feed 
on bacteria or algae occur in about 14% of the wet, organic matter-rich soils across 
the Dry Valley landscape (Freckman and Virginia 1997). In contrast to those that 
colonize plant-dominated soils, Dry Valley taxa rarely coexist as a community or 
more complex food web, and competitive interactions can be limited (Hogg et al. 
2006). Extreme soils can also be characterized by an absence of consumer popula- 
tions and their predators. About 60% of the soils in the Dry Valleys lack nematodes 
and about 50% of soils in Ellsworth Land, Antarctica, and areas in the Atacama 
Desert lack soil mesofauna (Convey and Mclnnes 2005; Freckman and Virginia 
1997; Warren-Rhodes et al. 2006). Whether these unsuitable soil habitats are due 
to soil geochemical and/or food source limitations, or else to other factors, is being 
studied (Poage et al. in press; Warren-Rhodes et al. 2006). 



76 D.H. Wall 

It is somewhat easier to clarify the food sources within an extreme soil food web, 
and thus the role of a species in the ecosystem, than it is in highly diverse soils. All 
the consumers, micro- and/or mesofauna are usually known at the species level in 
an extreme soil ecosystem. For example, using stable isotopes, Bokhorst et al. 
(2007) showed that a polar collembolan species feeds preferentially on lichens and 
algae, rather than moss. Less is known about faunal species feeding on a selective 
bacterial species, particularly for extreme soils where microbial diversity is appear- 
ing to be higher than previously reported (Aislabie et al. 2006; Barrett et al. 2006; 
Cowan and Tow 2004). Nevertheless, compared to the study of more diverse food 
webs, analysis of the extreme soil food webs is particularly useful to reveal food web 
architecture, the role of the species in the ecosystem, and the degree of overlap in 
geographic species range for soil fauna. 

Food webs in nonextreme soils have high energy input from plants and algae, 
more trophic levels, and potentially hundreds of species in a functional group. 
Larger macrofauna prey on smaller mesofauna, and so on through the food web. 
Food webs are thus extremely complex: for example, the diversity of fungal feed- 
ing mite species in soils might range in the hundreds of species whereas in an 
extreme soil, there may be only a single species, if any. Resolving food sources for 
each species in a functional group for most soils is thus extremely difficult due to 
the high number of species. Instead, transfer of nutrients, for example, carbon, 
nitrogen, and phosphorus, through the soil food web can only be estimated based 
on abundance and biomass of invertebrates within the various functional groups. 

Because most functional groups have many species performing the same task or 
role in highly diverse soil ecosystems, it has been argued that there is considerable 
redundancy (Loreau and Thebault 2005). If a species were lost, another species 
would take its place and there would be little change in the ecosystem function. 
More recently, experiments (Heemsbergen et al. 2004; Roscher et al. 2004) suggest 
that functional diversity is more important to an ecosystem function than the 
number of species (see also Hunt and Wall 2002). This is not the case in low-diversity 
systems where both numbers of functional groups and species are low (e.g., a func- 
tional group is represented by one species) and, frequently, one species is key to a 
process (Wall 2007). Loss of one species could decrease an ecosystem process in 
an extreme soil. For example, a single nematode species in the Dry Valley soils, the 
bacterial feeder Scottnema lindsayae, is responsible for a disproportionate amount 
of soil carbon turnover, about 5-7% (Barrett et al. unpublished), such level of activ- 
ity being unachievable in temperate ecosystems with their highly diverse and 
greater biomass (Schroter et al. 2003). As the Dry Valleys have cooled, populations 
of S. lindsayae have declined with as yet unknown implications for carbon cycling 
(Doran et al. 2002). 

Knowledge at an ecosystem level gained from studying simple food webs and 
individual species in extreme soils can be transferred to other terrestrial soils. 
Simply stated, microscopic species placed in a functional group may not be equal 
in their roles in an ecosystem process. Their roles may differ on temporal, spatial, 
physiological, nutritional, and other measurable scales, but may be masked by 
sheer numbers of species. Combined field and laboratory experimentation to clarify 



4 Biodiversity: Extracting Lessons from Extreme Soils 77 

food web interactions will enhance our ability to detect potential ecosystem effects 
involving loss of species or shift in composition of species (or functional groups). 
Synthesizing this information will enable us to better monitor how soil biodiversity 
is altered by global changes, to compare impacts across soil ecosystems, and to 
better formulate actions to assure long-term soil sustainability. 



4.5 Lesson Four: Soils Are Major Drivers of Biodiversity 

The geochemical component of extreme soils structures the diversity of life to a 
greater extent than the corresponding component in nonextreme soils, where biotic 
influences on soil organic matter and soil structure have masked effects of parent 
material. Many undisturbed extreme soils today reflect the past geologic history 
and parent material, and contribute to soil habitats that are highly heterogeneous at 
small and large spatial scales across the landscape. In ecosystems where plants are 
absent, for example, the hot hyperarid Atacama desert and the cold polar desert 
soils of the Dry Valleys, Antarctica, soils are relatively unchanged by centuries of 
biological (including human) activity and thus, the legacy of previous soil geo- 
chemistry patterns still remains. These deserts have extremely low water (<25mm 
mean annual rainfall for the Atacama, and <10cm rainfall equivalent for the Dry 
Valleys), low soil carbon, low organic matter, high pH, and high salinity (Barrett 
et al. 2004; Warren-Rhodes et al. 2006) compared to other ecosystems. As with 
other arid ecosystems, however, there is high spatial variability because soil chemi- 
cal (e.g., C, N, P, organic matter, pH, salinity) and physical factors (structure, tex- 
ture, soil type, pore space, bulk density) combine in varying proportions to form 
numerous habitats for organisms, which can range from suitable to poor (Barrett 
et al. 2004; Courtright et al. 2001; Wall and Virginia 1999). The soil geochemical 
heterogeneity affects the abundance of suitable habitats for life and contributes to 
patchily distributed fauna. Organisms, whether microbes, plants, or invertebrates, 
are limited by availability of soil resources at centimeter to kilometer scales 
(Ettema and Wardle 2002; Freckman and Virginia 1989; Poage et al. in press; 
Schlesinger et al. 1996; Wall and Virginia 1999; Warren-Rhodes et al. 2006). 

In this way, spatial segregation of species occurs in extreme soils without the 
influence of plant roots. For example, in the hyperarid hot Atacama Desert, absence 
of water determined the spatial scale distribution and presence of photo synthetic 
and heterotrophic bacteria (Warren-Rhodes et al. 2006). In cold desert Dry Valley 
soils, where vascular plants are lacking and average mean annual surface soil tem- 
peratures are -26°C, four nematode species are distributed across the landscape 
according to food sources and soil habitat geochemical characteristics (Barrett et al. 
2007; Porazinska et al. 2002; Treonis et al. 1999; Wall and Virginia 1999). S. lindsayae, 
the bacterial feeder that is widely distributed and has a greater abundance than the 
other nematode species, occurs in soils that are drier, saltier, and less organically 
rich. Another bacteria feeder, Plectus spp., is associated with soils that are moist, 
less saline, and with higher organic carbon; thus, this species rarely overlaps 



78 D.H. Wall 

geographically for food with S. lindsayae. Eudorylaimus antarcticus, the algal 
feeder (Wall 2007), is found in soil habitats that are moist and highly organic, but 
its highest abundance occurs in lake sediments and streams (Ayres et al. 2007; 
Treonis et al. 1999). Eudorylaimus and Plectus frequently co-occur, but infre- 
quently are found with S. lindsayae, or with a rarely found fourth species, 
Geomonhystera. sp. This soil food web in the Dry Valley soil ecosystem has no 
predators and is likely limited by physical constraints rather than species' competi- 
tion (Wall 2007). Other examples of simple food webs, broad niches, and spatial 
segregation have been seen in other extreme soils (Convey and Mclnnes 2005; 
Richard et al. 1994). These examples illustrate how the heterogeneity in soil habi- 
tats alone can be a major driver of local biogeographical patterns. 

Globally, geographical patterns of soil biodiversity are driven primarily by cli- 
mate and vegetation with soil heterogeneity having a variable role in determining 
biodiversity across spatial scales (Ettema and Wardle 2002). In younger soil systems, 
the soil biota, including plant roots, have contributed to the organic matter, total 
carbon, and soil structure by the formation of soil pores and channels. Thus, younger 
soils are subject to variation in biological, physical, and chemical alterations across 
shorter temporal and spatial scales than polar deserts, which tend to be older 
(Young and Crawford 2004). There are, however, examples of plant-dominated 
ecosystems where the soil substratum may be a stronger driver of belowground 
biogeographical patterns (Ettema and Wardle 2002). Fierer and Jackson (2006) 
show the influence of one soil factor, soil pH, as a predictor of soil microbial diver- 
sity across ecosystem types in North and South America. However, pH did not 
explain distribution of hypolithic soil bacteria in the Atacama Desert (Warren- 
Rhodes et al. 2006). In Arctic soils, where plants occur, variation in soil moisture was 
a major determinant of C0 2 respiration, which represents an overall measure of soil 
biotic metabolism (Sjogersten et al. 2006). 

Globally, the aboveground distribution patterns of animal and plant diversity 
generally follow the latitudinal gradient hypotheses (Gaston 1996) of increasing 
species diversity from the poles to the tropics (Willig et al. 2003). The question of 
whether microbes are everywhere globally (Fenchel and Finlay 2004; Finlay 2002), 
or instead have spatial biogeography such as a latitudinal gradient, has resurged as 
a scientific debate and spawned research to examine constraints to dispersal and 
colonization for microbes less than 500 |im in addition to organisms of larger size 
(Fierer and Jackson 2006; Hughes Martiny et al. 2006; Lawley et al. 2004). The 
discussion on microbes and biogeography has extended to include bacteria, Archea, 
and some Eukarya (e.g., unicellular algae, Protozoa). In extreme soils, recent stud- 
ies support unique organisms (Smith et al. 2006; Warren-Rhodes et al. 2006), but 
it is difficult to prove that these microbes are indeed unique (missing from other 
ecosystems) because of limited studies using similar molecular detection tech- 
niques or descriptions of soil habitat data. 

Termites are one of the few groups of soil invertebrates that appear to follow the 
latitudinal gradient pattern (Eggleton et al. 1996, 1995). Global biogeography for 
the majority of soil fauna, particularly the micro- and mesofauna is less well known 
(Bardgett et al. 2005; Hughes Martiny et al. 2006; Maraun et al. 2007) partially 



4 Biodiversity: Extracting Lessons from Extreme Soils 79 

because a greater proportion of soils have been sampled in temperate ecosystems 
(Bardgett 2005). One diverse group of soil microarthropods, oribatid mites, 
increases in diversity from boreal to temperate ecosystems but this trend does not 
extend to the tropics (Maraun et al. 2007), which may indicate a sampling problem. 
This problem has also been noted for global distribution patterns of soil nematodes 
(Bardgett 2005; Boag and Yeates 1998). 

Given the variation in global ecosystems, it is challenging to establish ecological 
hypotheses explaining patterns of biogeography for soil biodiversity (Willig et al. 
2003) or to determine if, at local regional or global scales, species-rich soils corre- 
spond to more productive ecosystems (Ettema and Wardle 2002; Young and 
Crawford 2004). Research in extreme ecosystems has already shown clearly that 
variation in soil geochemistry alone creates numerous soil habitats that are dis- 
tinctly different and suitable for some species, but not others. This information, 
combined with information on species dispersal and colonization and with further 
knowledge on other drivers (vegetation, climate) of soil biodiversity, should con- 
tribute to better predictions of global soil biogeography. 



4.6 Lesson Five: Global Changes Are Rapidly Changing Soils 

Global changes (climate change, atmospheric change, land use change, species extinc- 
tions, invasive species) are having an impact on soils at an increasingly rapid rate 
(Millennium Ecosystem Assessment 2005). Effects of soil degradation include loss of 
soil organic matter, erosion, salinisation, compaction, contamination, and sealing (Wall 
2004). Several international agreements address the irreversible loss of productive soils 
and the impact on biodiversity. As an example, the UN Convention to Combat 
Desertification was signed in 1997 by 178 nations to mitigate the effects of drought by 
implementation of action plans (UN Convention to Combat Desertification 1997). As 
recent as September 2006, the European Commission adopted a strategy specifically for 
soil protection by the EU (European Commission on Soil Protection 2006). These and 
other agreements have as a basis the knowledge of the benefits to humans (called eco- 
system services, see Section 4.1) provided by soils. The understanding that soil life is 
critically important for provision of ecosystem services is less well accepted as a basis 
for policy decisions than is the notion of the role of physical degradation (Wall 2004). 
Additionally, less attention has been drawn to extreme soils, with the exception perhaps 
of those organisms living in chemically impacted soils (see Chapters 15 and 16), 
partially because of the magnitude of the ecosystem change occurring to many species 
and habitats worldwide and because extreme soils do not produce crops (Alley et al. 
2007). Can knowledge of biodiversity in extreme soil ecosystems apply elsewhere, 
when globally there is accumulating evidence that soil functional composition and 
some soil species are being altered by global changes (Swift et al. 1998; Wardle et al. 
2004; Wolters et al. 2000)? 

In extreme soils, global changes may homogenize habitat ranges of species, with 
differing effects on these systems. Rapid environmental climate change could alter 



80 D.H. Wall 

species distribution as influenced by habitat requirements, physiological tolerances, 
and life histories. For example, warming in extremely cold ecosystems might 
increase soil moisture levels across large spatial scales and blend soil habitat chem- 
istry by affecting decomposition rates and the amount of carbon in the soil, primary 
production, and salinity levels (Wall 2007). In ecosystems with extreme drought 
events, soils might have reduced heterogeneity in chemical and physical properties 
due to high wind erosion. Other global changes in extreme soil systems, such as 
land use change (resulting from increased human activity) and increased incidence 
of invasive species, could also alter the food web interactions and relative sta- 
bility of extreme soils. Thus, the present habitats that specify species range could be 
altered significantly with consequent changes in species composition and geographic 
distributions and cascading effects on ecosystem processes across the landscape 
(Wall 2007). 

Little is known about the effect of global changes at the individual species level 
for most soil systems (Convey and Mclnnes 2005; Doran et al. 2002). Evidence from 
the study of extreme soil biodiversity suggests that global change effects will differ 
with species and ecosystem, and that even species with broad niches can be vulnera- 
ble (Barrett et al. unpublished). Thus, in extreme but also in more diverse soils, it will 
be important to quantify which, if any, species are key to an ecosystem process or 
ecosystem service and whether they are vulnerable to the many global changes. 



4.7 Conclusions 

Extreme soils may initially appear to be vastly different from each other and from 
the rest of the world's soils. There are several unique features of extreme soils: their 
lack of easily detectable life, reduced number of mesofaunal species within a func- 
tional group, fewer trophic levels, less complex food webs, lack of small-scale 
geographic overlap of species within functional groups, marked periodicity of 
activity, and food selectivity by invertebrate species (Convey and Mclnnes 2005; 
Porazinska et al. 2002; Treonis et al. 1999; Wall 2007). Collectively, the study of 
extreme soil habitats has revealed information on their biodiversity and on species 
interactions that are difficult to examine in nonextreme soils. Although much is 
yet to be learned, there is now sufficient evidence that species diversity in extreme 
soils has similarities to soil biodiversity elsewhere. Researchers interested in both 
extreme and nonextreme soils have used some species' traits, revealed at the genetic, 
population, community, or ecosystem levels, to obtain quantitative measurements 
of biodiversity. All of these criteria for biodiversity estimation are compatible 
with the Convention on Biological Diversity definition of biodiversity mentioned 
earlier, which encompasses variability from species to landscapes levels (Convention 
on Biological Diversity 2004). 

Extreme soil ecosystems are more than model systems or microcosms. The bio- 
diversity found in extreme soil environments is an integral part of the diversity of 
terrestrial surfaces. The extreme soil ecosystems are not anomalies; they are local 



4 Biodiversity: Extracting Lessons from Extreme Soils 81 

to relatively large terrestrial ecosystems with a range of life forms, albeit belonging to 
relatively few species, having several types of life histories and extraordinary 
physiological adaptations. For this reason alone, extreme soil habitats are worthy 
of conservation and protection. Kareiva and Marvier (2003) argued that ecosystems 
with fewer species, rather than those with high diversity, could be considered 
higher priorities for conservation. Extreme soils are also critically valuable as indi- 
cators of global changes likely to affect other soil systems. Our challenge is to 
define these habitats in more detail, to quantify their contribution to ecosystem 
processes and services, and to establish the relevance of biodiversity in extreme 
soils for sustaining life in all terrestrial soils. 



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Chapter 5 

Halophilic and Halotolerant Micro-Organisms 

from Soils 



Antonio Ventosa(K), Encarnacion Mellado, Cristina Sanchez-Porro, 
and M. Carmen Marquez 



5.1 Introduction 

Hypersaline environments are extreme habitats in which several other factors, in 
addition to high salt content, may limit the growth of organisms. These additional 
factors include temperature, pH, pressure, oxygen, nutrient availability, and solar 
radiations (Rodriguez- Valera 1988). Hypersaline environments comprise hyper- 
saline waters and soils. Hypersaline waters are defined as those environments that 
have higher concentrations of salts than seawater (Rodriguez- Valera 1988). 
However, depending on their origin, the salt composition may differ from that 
of seawater and on that basis hypersaline water habitats are categorized as thalas- 
sohaline, when the relative amounts of the different inorganic salts are approxi- 
mately equal to those present in seawater, or as athalassohaline, if the proportions 
of the different salts are markedly different from those of seawater. The later 
environments are more heterogeneous and may have very different origins. 
Examples of thalassohaline water habitats, which are typically chroride types, are 
the Great Salt Lake or the solar salterns used for the industrial production of 
marine salt by evaporation of seawater; among the athalassohaline waters are 
the Dead Sea, the Wadi Natrun, Lake Magadi, and several other soda lakes. In 
contrast to the hypersaline waters, the hypersaline soils are not well defined and 
in fact there is no clear definition of a saline or hypersaline soil. They are widely 
represented in our planet. Because most soils contain small amounts of soluble 
salts, a soil would be considered as hypersaline when its salt concentration is 
above a certain threshold (Rodriguez- Valera 1988). According to Kaurichev 
(1980), soils containing more than 0.2% (w/v) soluble salt should be considered 
as saline soils. 

Micro-organisms show quite different responses to salt. According to the 
particular salt concentration required for their optimal growth, several physiological 
groups of micro-organisms are considered: (i) nonhalophiles require less than 1% 



Antonio Ventosa 

Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 

41012 Sevilla, Spain 

e-mail: ventosa@us.es 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 87 

© Springer- Verlag Berlin Heidelberg 2008 



88 A. Ventosa et al. 

NaCl; (ii) halotolerant are nonhalophilic micro-organisms that can tolerate high salt 
concentrations, in some cases up to 25% NaCl; (iii) slight halophiles grow best in 
media with 1 to 3% NaCl; (iv) moderate halophiles grow optimally in media with 
3 to 15 % NaCl; and finally, (v) extreme halophiles grow best in media containing 
15 to 25% NaCl and are able to grow even at saturated salt concentrations (Kushner 
and Kamekura 1988). The organisms most commonly found in environments with 
high salt concentrations are the moderately halophilic bacteria and the extremely 
halophilic bacteria and archaea; however, halotolerant micro-organisms may be 
also present, although it is considered that they play a minor role in these environ- 
ments (Rodriguez- Valera 1988). 

In this chapter, we review the extremely and moderately halophilic micro-organ- 
isms that have been isolated from soils, as well as their ecological distribution. In 
addition, we present some current or envisioned biotechnological or industrial 
applications of these interesting micro-organisms. 



5.2 Microbial Diversity 

Compared to hypersaline aquatic habitats, very little information exists thus far 
regarding the diversity of halophilic and halotolerant micro-organisms isolated 
from saline soils, and much work needs to be done on this subject. Pioneering stud- 
ies on the microbial diversity in saline soils were carried out in the 1980s using 
conventional procedures and plate count methods. In 1982, Quesada and co-work- 
ers studied a hypersaline soil located in an abandoned multipond saltern, near the 
Mediterranean coast in Alicante, Spain. This kind of saltern provides a range of salt 
ponds with different salinities, ranging from salt content equivalent to that of sea- 
water up to sodium chloride saturation, and evaporation of the water in the ponds 
produces soils with a wide range of salt concentrations. The soil studied had NaCl 
concentrations which ranged from 5.0 to 10.7%; the majority of the organisms iso- 
lated from this soil were halophilic bacteria with optimal growth at salt concentra- 
tion between 5 and 15% and interestingly most of them were able to grow at 0.9% 
salt. This fact contrasts with the halo-dependence shown by most halophilic bacte- 
ria isolated from hypersaline waters or salted food, which in general have higher 
minimal salt requirements. 

One possible explanation for this phenomenon is the heterogeneity of the soil habi- 
tats, where the salinity can change markedly in space and time. As a consequence of 
this heterogeneity, excessively specialized organisms may periodically be eliminated 
and the euryhaline types more consistently favoured. Most isolates were Gram- 
negative rods (46.6%) that were identified as members of the genera Pseudomonas 
(22%), Alcaligenes (11%), Vibrio (currently Salinivibrio) (3%), Flavobacterium (3%), 
and Acinetobacter (1%). Most of the isolates assigned in this early study to the genera 
Pseudomonas, Alcaligenes, and Flavobacterium can probably be considered members 
of the current genus Halomonas. Gram-positive rods and Gram-positive cocci repre- 
sented 35.9% and 17.5% of the isolates, respectively, and were assigned to the genera 



5 Halophilic and Halotolerant Micro-Organisms from Soils 89 

Bacillus (19%), Micrococcus (currently Nesterenkonia) (8%), Arthrobacter (6%), 
Planococcus (currently Marinococcus) (5%), Staphylococcus (3%), Corynebacterium 
(2%), Brevibacterium (1%), Nocardia (1%), and Actinomyces (1%). 

A very low proportion of extremely halophilic archaea (1%) was isolated from 
this soil, probably due to an insufficient incubation period. They were assigned to 
the genus Halobacterium and their presence in this soil suggested the existence of 
local microsites with sufficiently high salt concentrations to allow the growth of 
halophilic Archaea. This study showed that the taxonomic groups found are the 
ones that also predominate in nonsaline soils; however, the microbial community 
structure in the saline soils studied is very different from that of communities in 
hypersaline waters, where the genera Salinivibrio and Halomonas are more fre- 
quently isolated (Ventosa et al. 1982, 1998; Marquez et al. 1987). 

Later studies showed that Gram-positive micro-organisms are extensively rep- 
resented in saline soils. Ventosa et al. (1983) isolated a group of Gram-positive 
halophilic cocci from the saline soil near Alicante mentioned before, all of them 
being moderate halophiles that grew between 2-5% and 25-30% total salts and 
optimally in media with 10-15% total salts. They were assigned to the species 
Planococcus (Marinococcus) halophilus and Sporosarcina (Halobacillus) halophila. 
In this study, no correlation was found between the isolation habitat or the salt range 
in which growth occurred and the taxonomic affiliation of the isolated strains. 
Garabito et al. (1998) isolated and studied 71 halotolerant Gram-positive endospore- 
forming rods from saline soils and sediments of salterns located in different areas of 
Spain (Huelva, Cadiz, Sevilla, and Mallorca). These isolates were tentatively 
assigned to the genus Bacillus, and the majority of them were classified as extremely 
halotolerant micro-organisms, being able to grow in most cases in up to 20 or 25% 
salt. It was not possible to determine if these isolates were normal inhabitants of the 
environments studied, although, because they showed a very wide salt growth range, 
their presence as inhabitants of hypersaline environments is quite probable. 

Members of the genus Bacillus have been isolated from almost all natural 
habitats and from many other sources, owing to the ability of their spores to be 
transported and their remarkable capacity for resistance and dormancy, which 
allows them to survive in unfavorable habitats for long periods (Claus and Berkeley 
1986). Extremely halotolerant cocci with similar characteristics to those described 
for the species Micrococcus halobius (currently Nesterenkonia halobia) were iso- 
lated from saline soils in different regions of the Antarctic continent (Nicolaus 
et al. 1992). Saline soils are a feature of Antarctic regions and halotolerant cocci 
had been isolated from this continent before (Miller et al. 1983; Miller and Leschine 
1984); however, this is the first study where extremely halotolerant micrococci 
were reported. 

During the last decade, advances in techniques for studying environmental 
microbiology have changed our views of the microbial communities in soils. 
Application of molecular biological methods has revealed an astonishing level of 
microbial diversity in the biosphere, only a small proportion of which has been 
assessed through cultivation (DeLong and Pace 2001; Rappe and Giovannoni 2003). 
Soils and sediments are among the most diverse microbial ecosystems and are 



90 A. Ventosa et al. 

estimated to contain an order of magnitude more of different prokaryotic "species" 
than do aquatic environments (Curtis et al. 2002; Torsvik et al. 2002); perhaps this 
is due to the higher spatial heterogeneity present in soil environments. 

Using a cultivation-independent approach based on 16S rRNA gene analysis, 
Walsh et al. (2005) examined the archaeal diversity along a soil salinity gradient 
prone to frequent disturbance in the form of salinity fluctuations at Salt Spring, in 
British Columbia, Canada. Soil samples were collected from three sites along a 
salinity gradient ranging from 7 to 18% NaCl. The archaeal richness across the 
different sites studied was similar; however, a significant shift in archaeal com- 
munity composition was found along the salinity gradient. The haloarchaea (70% 
of the clones) were the most commonly sampled group in this study, followed by 
other Euryarchaeota (26%), Crenarchaeota (3%), and halophilic methanogens 
(1%). It was observed that an increase in the salt concentration of the soils was 
accompanied by an increase in haloarchaeal diversity and a corresponding 
decrease in the other archaeal groups. Over 130 unique haloarchaeal sequences, 
representing 38 ribogroups, were recovered; these data suggest that the haloar- 
chaeal community at Salt Spring is exceptionally diverse in comparison with other 
previously characterized haloarchaeal communities from solar salterns (Benlloch 
et al. 2002). The haloarchaeal 16S rRNA gene sequences recovered from Salt 
Spring soil were comprised of many lineages distributed throughout the haloar- 
chaea, and were related to those of the genera Halorubrum, Natrinema, 
Natronorubrum, Haloterrigena, and Natronococcus. 

The recovery of such a diversity of haloarchaea was unexpected as the majority 
of known haloarchaea undergo lysis at NaCl concentrations below 9% NaCl (Grant 
et al. 2001) and the salinity of Salt Spring appears to be below this threshold level 
for a considerable period of the year. However, Salt Spring haloarchaea were not 
closely related to known low salt-adapted or tolerant species (Rodriguez-Valera 
et al. 1979; Munson et al. 1997; Purdy et al. 2004; Elshahed et al. 2004), suggesting 
they may be frequently faced with local mortality as a result of frequent declines in 
soil salinity. 

Very recently, Jiang et al. (2006) employed culture-independent (16S rRNA gene 
analysis) and culture-dependent microbiological techniques to assess microbial diver- 
sity and abundance in Lake Chaka, a hypersaline lake in northwestern China. Microbial 
abundance in the sediments of this lake ranged between 10 8 cells/g at the water- 
sediment interface and 10 7 cells/g at a sediment depth of 42 cm. The isolates from the 
sediment showed a halotolerant nature with an optimum salinity of 5% NaCl or lower, 
consistent with the salinity in the sediment. With respect to the bacterial diversity 
present in the sediment samples, the majority of the sequences obtained were affiliated 
with the Firmicutes (low-GC Gram-positive bacteria). This was in contrast with popu- 
lations from the water samples, dominated by halophilic bacteria with clone sequences 
related to the Bacteroides group. Similar differences were also observed in the archaeal 
community, with all archaeal clone sequences in the lake water belonging to the order 
Halobacteriales, whereas most sequences in the sediment libraries were related to 
Euryarchaeota group III sequences, previously found in a diverse range of environ- 
ments including methanogenic soils and sediments. A small percentage of sequences 



5 Halophilic and Halotolerant Micro-Organisms from Soils 91 

from the sediment libraries was related to the Crenarchaeota group. All sequences 
in Euryarchaeota group III were preliminarily assigned to Thermoplasmales; however, 
definitive identification was not possible because pure isolates were not available. It is 
interesting to point out that sequences belonging to Thermoplasmales have been 
reported to be present in saline soils (Walsh et al. 2005). 



5.3 Halophilic Micro-Organisms from Soils 

Most halophilic and halotolerant micro-organisms isolated and characterized in 
detail from saline or hypersaline soils are heterotrophic bacteria, but some haloar- 
chaea have also been described. Here, we review the features of the halophilic 
archaea and bacteria isolated from soils. 



5.3.1 Extremely Halophilic Archaea 

With the exception of some halophilic methanogens, the extremely halophilic archaea 
belong to the haloarchaea, members of the family Halobacteriaceae within the 
Euryarchaeota. They are aerobic micro-organisms producing red to pink carotenoid 
pigments and have characteristic archaeal features, such as membranes containing 
ether-linked isoprenoid chains; their most typical feature is an absolute NaCl require- 
ment, inasmuch as most species require at least 9% NaCl for growth and grow opti- 
mally in media with 20-25% NaCl (Grant et al. 2001). However, recent studies on 
coastal salt-marsh sediments have shown that some isolates are able to grow optimally 
at 10% NaCl and even grow slowly at 2.5% NaCl (Purdy et al. 2004). Currently, the 
haloarchaea are represented by more than 60 species grouped in 22 genera (Ventosa 
2006). They are common inhabitants of hypersaline environments, and most species 
have been isolated from aquatic habitats such as salt lakes, soda lakes, or salterns, as 
well as from salted foods and subterranean salt deposits (Grant et al. 2001). 

Only a few haloarchaeal species have been isolated from saline soils: they are 
Haloarcula argentinensis and Haloarcula mukohatae, two species isolated in 
Argentina (Ihara et al. 1997); Haloarcula japonica, isolated from saltern soil in 
Japan (Takashina et al. 1990); Halorubrum distributum (formerly Halobacterium 
distributes), isolated from alkaline soils (Zvyagintseva and Tarasov 1987; Oren and 
Ventosa 1996); and Haloterrigena turkmenica (formerly described as Halococcus 
turkmenicus), isolated from a sulfate saline soil in Turkmenistan (Zvyagintseva and 
Tarasov 1987; Ventosa et al. 1999). In addition to these well-characterized species, 
several other haloarchaea have been isolated from saline soils, such as Haloferax 
sp. strain D1227, isolated from a soil contaminated with saline oil brine near Grand 
Rapids, Michigan, USA. This extremely halophilic organism is able to utilize a 
variety of aromatic compounds; in particular, the degradation of 3-phenylpropionic 
acid by this haloarchaeon has been studied (Fu and Oriel 1998, 1999). 



92 



A. Ventosa et al. 



5.3.2 Moderately Halophilic Bacteria 

Most moderately halophilic bacteria isolated and described from soils are hetero- 
trophic bacteria. In contrast to hypersaline water habitats, from which predominantly 
Gram-negative species were obtained, saline or hypersaline soils have yielded many 
Gram-positive species, and these have been characterized taxonomically. The micro- 
biota of hypersaline soils are more similar to those of nonsaline soils than to the 
microbiota from hypersaline waters. This suggests that general features of the envi- 
ronments are more important in determining the microbiota in a particular habitat 
than are individual factors such as high salinity (Quesada et al. 1983). Lists of val- 
idly described halophilic species names and their isolation site are presented here for 
Gram-positive (Table 5.1) and Gram-negative (Table 5.2) species. 



Table 5.1 Selected 
hypersaline soils 



moderately halophilic Gram-positive species isolated from saline or 







NaCl Range and 




Species 


Isolation Source 


Optimum (%) 


Reference 


Actinopolyspora 


Soil sample in Iraq 


5-20 (10-15) 


Ruan et al. 


iraqiensis 






(1994) 


Actinopolyspora 


Soil sample obtained from Death 


5-30 (10-15) 


Yoshida et al. 


mortivallis 


Valley, CA, USA 




(1991) 


Alkalibacillus 


Alkaline, highly saline mud from 


5-20 (10) 


Jeon et al. 


haloalkaliphilus 


Wadi Natrun, Egypt 




(2005b) 


Alkalibacillus 


Soil sediment from a salt lake in 


5-20% (10-12%) 


Jeon et al. 


salilacus 


Xinjiang Province, China 




(2005b) 


Bacillus krulwichiae 


Soil from Tsukuba, Ibaraki, 


0-14 


Yumoto et al. 




Japan 




(2003) 


Bacillus oshimensis 


Soil from Oshymanbe, Oshima, 


0-20 (7) 


Yumoto et al. 




Hokkaido, Japan 




(2005) 


Bacillus 


Rhizosphere of the perennial 


0-15 


Olivera et al. 


patagoniensis 


shrub Atriplex lampa in 
north-eastern Patagonia, 
Argentina 




(2005) 


Desulfobacter 


Sediment of Great Salt Lake, 


0.5-13 (1.2) 


Brandt and 


halotolerans 


Utah, USA 




Ingvorsen 
(1997) 


Filobacillus 


Beach sediment from Palaeochori 


2-23 (8-14) 


Schlesner et al. 


milosensis 


Bay, Milos, Greece 




(2001) 


Halobacillus 


Salt marsh and saline soils 


2-15 (3-5) 


Spring et al. 


halophilus 






(1996); 
Ventosa et al. 
(1983) 


Halobacillus 


Saline soil of the Karaj region, 


1-24 (10) 


Amoozegar et al. 


karajensis 


Iran 




(2003b) 


Lentibacillus 


Saline sediment of Xinjiang 


1-20(12-14) 


Jeon et al. 


salarius 


Province, China 




(2005a) 



(continued) 



5 Halophilic and Halotolerant Micro-Organisms from Soils 
Table 5.1 (continued) 



93 







NaCl Range and 




Species 


Isolation Source 


Optimum (%) 


Reference 


Lentibacillus 


Salt field in Korea 


2-23 (4-8) 


Yoon et al. 


salicampi 






(2002) 


Marinococcus 


Saline soil from Alicante and 


1-20 (15) 


Hao et al. 


halophilus 


Cadiz, Spain 




(1984); 
Marquez 
et al. (1992) 


Marinococcus 


Saline soil in Qinghai, north-west 


0-25 (10) 


Li et al. (2005c) 


halotolerans 


China 






Micro bacterium 


Soil sediment of Qinghai 


0-15 (5) 


Li et al. (2005a) 


halotolerans 


Province, China 






Nesterenkonia 


Hypersaline soil from Xinjiang 


0-25 


Li et al. (2004b) 


halotolerans 


Province, China 






Nesterenkonia 


Hypersaline soil from Xinjiang 


0-25 


Li et al. (2004b) 


xinjiangensis 


Province, China 






Nocardiopsis 


Saline sediment from Xinjiang 


0-18 (5-8) 


Li et al. (2006) 


baichengensis 


Province, China 






Nocardiopsis 


Saline sediment from Xinjiang 


0-18 (5-8) 


Li et al. (2006) 


chromatogenes 


Province, China 






Nocardiopsis gilva 


Saline sediment from Xinjiang 
Province, China 


0-18 (5-8) 


Li et al. (2006) 


Nocardiopsis 


Saline soil from Iraq 


3-20 (5-15) 


Al-Tai and 


halophila 






Ruam, (1994) 


Nocardiopsis 


Salt marsh soil from Kuwait 


0-15 (10) 


Al-Zarban et al. 


halotolerans 






(2002a) 


Nocardiopsis 


Saline sediment from Xinjiang 


0-18 (5-8) 


Li et al. (2006) 


rhodophaea 


Province, China 






Nocardiopsis rosea 


Saline soil from Xinjiang 
Province, China 


0-18 (5-8) 


Li et al. (2006) 


Nocardiopsis salina 


Saline soil from Xinjiang 
Province, China 


3-20 (10) 


Li et al. (2004a) 


Nocardiopsis 


Saline soil from Xinjiang 


10 


Li et al. (2003a) 


xinjiangensis 


Province, China 






Prauserella alba 


Saline soil from Xinjiang 
Province, China 


0-25 (10-15) 


Li et al. (2003c) 


Prauserella 


Saline soil from Xinjiang 


5-25 (10-15) 


Li et al. (2003c) 


halophila 


Province, China 






Saccharomonospora 


Marsh soil in Kuwait 


10-30 (10) 


Al-Zarbam et al. 


halophila 






(2002b) 


Saccharomonospora 


Soil from Xinjiang Province, 


5-20 (10) 


Li et al. (2003b) 


paurometabolica 


China 






Salinicocccus 


Saline soil from Alicante and 


0.5-25 (10) 


Marquez et al. 


hispanicus 


Cadiz, Spain 




(1990) 


Sporohalobacter 


Dead Sea sediment 


6-12 (8.7) 


Oren (1983) 


lortetii 








Streptomonospora 


Soil from Xinjiang Province, 


5-25 (10-15) 


Li et al. (2003d) 


alba 


China 






Streptomonospora 


Soil from Xinjiang Province, 


15 


Cui et al. (2001) 


salina 


China 







(continued) 



94 



A. Ventosa et al. 



Table 5.1 (continued) 



Species 



Isolation Source 



NaCl Range and 
Optimum (%) 



Reference 



Tenuibacillus 

multivorans 
Thalassobacillus 

devorans 
Virgibacillus 

koreensis 
Virgibacillus 

salexigens 



Soil from Xinjiang Province, 

China 
Saline soil in South Spain 



1-20 (5-8) 
0.5-20 (7.5-10) 



Salt field near Taean-Gun on the 0.5-20 (5-10) 

Yellow Sea in Korea 
Soil from Huelva, Cadiz, Sevilla, 7-20 (8- 10) 

and Mallorca, Spain 



Ren and Zhou 

(2005) 
Garcia et al. 

(2005a) 
Lee et al. (2006) 

Garabito et al. 
(1997) 



Table 5.2 Selected moderately halophilic Gram-negative species isolated from saline or hypersa- 
line soils 







NaCl Range and 




Species 


Isolation Source 


Optimum (%) 


Reference 


Halanaerobacter 


Salt ponds in Camargue, France 


5-30 (14-15) 


Moune et al. 


salinarius 






(1999) 


Halanaerobium 


Sediment from Great Salt 


2.5-25 (10) 


Tsai et al. (1995) 


alcaliphilum 


Lake, Utah, USA 






Halanaerobium 


Sediment of Retba Lake, 


7.5-34 (18-20) 


Cayol et al. 


lacusrosei 


Senegal 




(1995) 


Halanaerobium 


Sediment of Great Salt Lake. 


2-30 (20) 


Zeikus et al. 


praevalens 


Utah, USA 




(1983) 


Halanaerobium 


Sediments of Retba Lake, 


5-25 (7.5-12.5) 


Cayol et al. 


saccharolytica 


Senegal 




(1994b) 


subsp. senegalensis 








Halomonas 


Soil from Fuente de Piedra. 


0.5-15 (7.5) 


Martinez-Canovas 


anticariensis 


Malaga, Spain 




et al. (2004a) 


Halomonas 


Soil around the lake Laguna 


0-25 (5) 


Quillaguaman 


boliviensis 


Colorada, Bolivia 




et al. (2004) 


Halomonas 


Soil sample collected from a 


1-25 (8.7) 


Mormile et al. 


campisalis 


dry salt flat south of Alkali 
Lake, USA 




(1999) 


Halomonas 


Hypersaline soil in Alicante, 


0.5-30 (7.5) 


Quesada et al 


eurihalina 


Spain 




(1990); 
Mellado et al. 
(1995) 


Halomonas 


Hypersaline soil located near 


2-30 (7.5) 


Quesada et al. 


halophila 


Alicante, Spain 




(1984) 


Halomonas maura 


Soil from a solar saltern at 


1-15 (7.5-10) 


Bouchotroch 




Asilah, Morocco 




et al. (2001) 


Halomonas 


Saline soil from Isla Cristina, 


1.5-30(7.5-10) 


Garcia et al. 


organivorans 


Huelva, Spain 




(2004) 


Halomonas salina 


Saline soils located near 


2.5-20 (5) 


Valderrama et al. 




Alicante, Spain 




(1991) 


Halomonas 


Saline soil in Jaen, Spain 


3-15 (6-9) 


Martinez-Canovas 


ventosae 






et al. (2004b) 



(continued) 



5 Halophilic and Halotolerant Micro-Organisms from Soils 
Table 5.2 (continued) 



95 







NaCl Range and 




Species 


Isolation Source 


Optimum (%) 


Reference 


Halothermotrix 


Sediment of a Tunisian salt 


4-20 (5-10) 


Cayol et al. 


orenii 


lake 




(1994) 


Marinobacter 


Sediment collected from 


1-15 


Gorshkova et al. 


excellens 


Chazhman Bay, Sea of 
Japan 




(2003) 


Marinobacter 


Sea sand in Pohang, Korea 


0.5-20 


Kim et al. (2006) 


koreensis 








Marinobacter 


Saline soil from Cadiz, Spain 


1-15 (7.5) 


Martin et al. 


lipolyiticus 






(2003) 


Marinobacter 


Marine coastal sediment from 


0.5-18 


Romanenko et al. 


sediminum 


Peter the Great Bay, Sea of 
Japan 




(2005) 


Natroniella 


Mud from the soda Lake 


10-26 (12-15) 


Zhilina et al. 


acetigena 


Magadi, Kenya 




(1999) 


Orenia salinaria 


Salt ponds in salterns in 


2-25 (5-10) 


Moune et al. 




Camargue, France 




(2000) 


Palleronia 


Hypersaline soil bordering a 


0.5-15 (5) 


Martinez-Checa 


marisminoris 


solar saltern in Murcia, 
Spain 




et al. (2005) 


Salipiger mucosus 


Hypersaline soil from a solar 


0.5-20 (3-6) 


Martinez-Canovas 




saltern in Calblanche, 




et al. (2004c) 




Murcia, Spain 







5.3.2.1 Gram-Positive Bacteria 



In nature, salinity often goes together with alkalinity. Several alkaliphilic Bacillus 
species have been identified to date (Vedder 1934; Spanka and Fritze 1993; Nielsen 
et al. 1995; Agnew et al. 1995; Fritze, 1996; Switzer et al. 1998; Yumoto et al. 
2003; Olivera et al. 2005). Some of these have been isolated from soil samples and 
show halophilic characteristics. Bacillus krulwichiae (Yumoto et al. 2003), iso- 
lated in Tsukuba, Japan, is a facultatively anaerobic, straight rod with peritrichous 
flagella that produces ellipsoidal spores and utilizes benzoate or m-hydroxybenzoate 
as the sole carbon source. Bacillus patagoniensis (Olivera et al. 2005) was isolated 
from the rhizosphere of the perennial shrub Atriplex lampa in north-eastern 
Patagonia. Finally, Bacillus oshimensis (Yumoto et al. 2005) is a halophilic nonmo- 
tile, facultatively alkaliphilic species. It exhibits a NaCl requirement for growth, and 
salt may be required for pH homeostasis, adaptation to an alkali environment, or 
energy production through the respiratory chain (Tokuda and Unemoto 1981, 1984) 
or ATPase function (Ueno et al. 2000). 

Some moderately halophilic, spore-forming, Gram-positive rods were originally 
assigned to the genus Bacillus, but have been reclassified within new genera by the 
application of molecular methods and improved phenotypic approaches (Heyndrickx 
et al. 1998; Yoon et al. 2001, 2004). Indeed, 16S rRNA sequence and chemotaxonomic 
analyses revealed the existence of several phylogenetically distinct lineages within 



96 A. Ventosa et al. 

the genus Bacillus (Ash et al. 1991; Nielsen et al. 1994). One example of such 
independent lineage is a group comprising the species Alkalibacillus haloalkaliphilus 
(formerly Bacillus haloalkaliphilus', Fritze, 1996; Jeon et al. 2005b) isolated from 
alkaline, high saline mud from Wadi Natrun, Egypt, and Alkalibacillus salilacus, iso- 
lated from soil sediment of a salt lake in China (Jeon et al. 2005b). 

Another example is the genus Virgibacillus. This genus was first proposed by 
Heyndrickx et al. (1998) based on polyphasic data and its description was later 
emended by Heyrman et al. (2003). Members of the genus Virgibacillus produce 
oval to ellipsoidal endospores, are Gram-positive motile rods, and have DNA G+C 
content ranging from 36 to 43 mol% (Heyrman et al. 2003). Currently, this genus 
comprises eight species, two of which are moderately halophilic and have been 
isolated from soil samples: Virgibacillus salexigens (Garabito et al. 1997; Heyrman 
et al 2003) and the recently described Virgibacillus koreensis (Lee et al. 2006). 

Several other aerobic or facultatively anaerobic, moderately halophilic, 
endo spore-forming, Gram-positive bacteria have been classified within genera 
related to Bacillus. Genera that include halophilic species isolated from soil sam- 
ples are Halobacillus, Filobacillus, Tenuibacillus, Lentibacillus, and Thalassobacillus. 
The genus Halobacillus is clearly differentiated from other related genera on the 
basis of its cell- wall peptidoglycan type; members of this genus have peptidoglycan 
based on L-Orn-D-Asp (Spring et al. 1996). The genus Filobacillus has peptidog- 
lycan based on L-Orn-D-Glu (Schlesner et al. 2001). Within these genera, the halo- 
philic species isolated from soils are: Halobacillus halophilus (Spring et al. 1996), 
Halobacillus karajensis (Amoozegar et al. 2003b), and Filobacillus milolensis 
(Schlesner et al. 2001). With respect to the genus Lentibacillus, two halophilic soil 
species are recognized, Lentibacillus salicampi isolated from a salt field in Korea 
(Yoon et al. 2002), and Lentibacillus salarius from a saline sediment in China (Jeon 
et al. 2005a). Finally, the genera Tenuibacillus and Thalassobacillus are comprised 
of only one species, Tenuibacillus multivorans isolated from a saline soil in Xin- 
jiang, China (Ren and Zhou 2005) and Thalassobacillus devorans, isolated from a 
saline soil in South Spain (Garcia et al. 2005a). All these genera belong to the family 
Bacillaceae, included in the phylogenetic group of the low GC Gram-positive 
bacteria, and are closely related. 

The genus Marinococcus was proposed in 1984 to accommodate two moderately 
halophilic species, Marinococcus halophilus and Marinococcus albus (Hao et al. 
1984). A third species, Marinococcus halotolerans , has been described recently 
(Li et al. 2005c). They are motile cocci that grow over a wide range of salt concen- 
trations and up to 20% NaCl, have m£S6>-diaminopimelic acid in their cell wall, a 
DNA G+C content ranging between 43.9 and 46.6 mol%, and MK-7 as the menaqui- 
none system. Of these species, M. halophilus is the most commonly isolated and it 
occurs in most hypersaline environments (Ventosa et al. 1983; Marquez et al. 
1992). M. halophilus and M. halotolerans were originally isolated from soil sam- 
ples located in Spain and China, respectively. Salinicoccus hispanicus (formerly 
Marinococcus hispanicus; Marquez et al. 1990; Ventosa et al. 1992) was isolated 
from a hypersaline soil located near Alicante (Spain). This species comprises 
Gram-positive, nonmotile, and non- spore-forming cocci. 



5 Halophilic and Halotolerant Micro-Organisms from Soils 97 

The genus Microbacterium was established by Orla- Jensen (1919) and emended 
by Collins et al. (1983). It comprises a diverse group of Gram-positive, non- 
spore-forming rods. More recently, this genus was emended again to unify the 
genera Microbacterium and Aureobacterium (Takeuchi and Hatano 1998). Members 
of the genus Microbacterium can be isolated from a wide range of habitats, including 
soil, water, plants, steep liquor, milk products, and humans. Microbacterium 
halotolerans, an aerobic, Gram-positive, nonmotile, non- spore-forming short rod, 
is the only halophilic species of this genus isolated from a soil sample, specifically 
from a saline soil in the west of China (Li et al. 2005a). 

The genus Micrococcus was first described more than 100 years ago, and since 
then its description has been revised several times. A phylogenetic and chemotaxo- 
nomic reanalysis of the genus Micrococcus resulted in the proposal of the genus 
Nesterenkonia (Stackebrandt et al. 1995) constituted by coccoid or short Gram- 
positive rods, non- spore-forming, chemo-organo trophic with strictly respiratory 
metabolism. Species of this genus are aerobic, catalase-positive, and moderately 
halophilic or halotolerant (Stackebrand et al. 1995; Collins et al. 2002; Li et al. 
2005b). Two species isolated from soil habitats are Nesterenkonia halotolerans and 
Nesterenkonia xinjiangensis (Li et al. 2004a). 

The presence and activity of dissimilatory sulfate-reducing bacteria in hypersaline 
environments have been reported by several investigators (Triiper 1969; Caumette 
1993; Oren 1988; Tardy-Jacquenod et al. 1996). Some of the highest sulfate reduction 
rates recorded have been measured in hypersaline microbial mats with salinities up to 
18-21% (Caumette et al. 1994). Active sulfate reduction has also been documented 
in hypersaline sediments (Zeikus 1983; Nissenbaum 1975; Skyring 1987). In 1983, 
Oren isolated a sulfate-reducing, anaerobic, halophilic, rod-shaped, endospore-forming 
bacterium from Dead Sea sediments. This bacterium was designated as Clostridium 
lortetii and later it was reclassified as Sporohalobacter lortetii (Oren 1983; Oren et 
al. 1987). The cells of this species are characterized by the production of gas vesicles, 
generally near the developing terminal endospore, and these vesicles remain attached 
to the mature endospore after degeneration of the vegetative cell. 

The genus Desulfobacter comprises nutritionally specialized sulfate reducers 
with acetate as their characteristic substrate, which is oxidized via a modified citric 
acid cycle (Hansen 1994; Moller et al. 1987). Desulfobacter halotolerans is the first 
acetate-oxidizing, sulfate-reducing, and halophilic bacterium to have been recog- 
nized. It was isolated from sediments of Great Salt Lake, Utah (Brandt and 
Ingvorsen 1997). This bacterium reduces sulfate, thiosulfate, or sulfite. Acetate, 
ethanol, and pyruvate are used as electron donors and carbon sources. 

The occurrence of actinomycetes in highly saline environments and the toler- 
ance of these organisms to high salt concentrations was first described by Tresner 
et al. (1968) and Gottlieb (1973). The family Nocardiopsaceae contains three gen- 
era, namely Nocardiopsis (Meyer 1976), Thermobifida (Zhang et al. 1998), and 
Streptomonospora (Cui et al. 2001). At present, the genus Nocardiopsis comprises 
19 validly published species names (Li et al. 2003a, 2004b; Al-Zarban et al. 2002a). 
These species comprise aerobic, Gram-positive, non-acid-fast, and nonmotile 
organisms. Originally, members of this genus had been isolated from mildewed 



98 A. Ventosa et al. 

grain (Brocq-Rosseau 1904), but the natural habitat of Nocardiop sis is soil. It has 
been reported to predominate in saline or alkaline soils (Tang et al. 2003) and sev- 
eral recognized species have been isolated from such sources (Al-Tai and Ruan 
1994; Chun et al. 2000; Al-Zarban et al. 2002a; Li et al. 2003a, 2004b). 

Some examples of moderately halophilic species of the genus Nocardiopsis 
isolated from soil samples are: Nocardiopsis halophila (Al-Tai and Ruan 1994), 
Nocardiopsis halotolerans (Al-Zarban et al. 2002a), and several species isolated 
during a taxonomic study of extremophilic actinomycetes from hypersaline soils 
in Xinjiang Province, China; these are Nocardiopsis xinjiangensis (Li et al. 2003a), 
Nocardiopsis salina (Li et al. 2004b), and the recently described species 
Nocardiopsis gilva, Nocardiopsis rosea, Nocardiopsis rhodophaea, Nocardiopsis 
chromato genes, and Nocardiopsis baichengensis (Li et al. 2006). The genus 
Streptomonospora is constituted by only two species, which are Gram-positive, 
aerobic organisms with branching hyphae that grow optimally in media with 15% 
NaCl: Streptomonospora alba and Streptomonospora salina, both of them isolated 
from the same sampling site in China (Cui et al. 2001; Li et al. 2003d). The name 
Streptomonospora was coined to indicate that species from this genus form two 
types of spores, including spores in short chains on the aerial mycelium and single 
spores with wrinkled surfaces on the substrate mycelium (Cui et al. 2001). 

The family Pseudonocardiaceae, which also belong to the order Actinomycetales, 
comprises many genera, three of which, Actinopolyspora, Saccharomonospora, and 
Prauserella, contain halophilic species isolated from saline soil samples. During 
the course of screening for antibiotics, a moderately halophilic actinomycete that 
produced nucleoside antibiotics was isolated from salty soil obtained from Death 
Valley, CA, USA. On the basis of its morphological and chemical properties, it was 
assigned to the genus Actinopolyspora as Actinopolyspora mortivallis (Yoshida et al. 
1991). Another species belonging to this genus is Actinopolyspora iraqiensis, 
which was obtained concomitantly with Nocardiopsis halophila, both of them 
originating from a saline soil from Iraq (Al-Tai and Ruan 1994; Ruan et al. 1994). 

The genus Saccharomonospora (Nonomura and Ohara 1971) includes actino- 
mycetes producing predominantly single spores on aerial hyphae. Saccharomonospora 
halophila was the first halophilic species of this genus, and was isolated from a 
marsh soil in Kuwait (Al-Zarban et al. 2002b). The studies on halophilic actino- 
mycetes carried out by Jiang and coworkers (Li et al. 2003a,b,c,d, 2004a, 2006; Cui 
et al. 2001) in hypersaline soils of the Xinjiang Province, China, led to the isolation 
of the novel species Saccharomonospora paurometabolica (Li et al. 2003b) as well 
as of two new halophilic species belonging to the genus Prauserella: Prauserella 
halophila and Prausella alba (Li et al. 2003c). 



5.3.2.2 Gram-Negative Bacteria 

Many Gram-negative, moderately halophilic, or halotolerant species are 
currently included in the family Halomonadaceae, which belongs to the Gamma- 
proteobacteria (Arahal and Ventosa 2005). This family includes three genera with 
halophilic species: Halomonas, Chromohalobacter, and Cobetia, plus two genera 



5 Halophilic and Halotolerant Micro-Organisms from Soils 99 

of nonhalophilic bacteria, Zymobacter and Carnimonas (Arahal et al. 2002a; Dobson 
and Franzmann 1996; Garriga et al. 1998; Okamoto et al. 1993; Yakimov et al. 1998; 
Ventosa et al. 1989). Among the genera that comprise this family, Halomonas covers 
the greatest number of species (more than 40) showing heterogeneous features. 
Based on the comparison of the 16S and 23S rRNA sequences, several phylogenetic 
groups have been delineated within this genus (Arahal et al. 2002b). It has also been 
noted that the genus Halomonas has an unusually wide range of DNA G+C content, 
from 52 to 68mol% (Arahal et al. 2002b). This genus comprises slightly or moderately 
halophilic, chemo-organotrophic, Gram-negative rods, which are widely distributed 
throughout hypersaline environments (Arahal and Ventosa 2005). 

Although the majority of them have been isolated from aquatic habitats, some 
species were isolated from soil samples: Halomonas halophila (formerly Deleya 
halophila; Quesada et al. 1984), Halomonas salina (formerly Deleya salina; 
Valderrama et al. 1991), and the recently described Halomonas organivorans, those 
three species originating from saline soil samples in Spain. Halomonas campisalis 
(Mormile et al. 1999) and Halomonas boliviensis (Quillaguaman et al. 2004) were 
described as alkaliphilic and alkalitolerant moderately halophilic bacteria, respec- 
tively, inasmuch as these bacteria are able to grow in media with pH values of about 
8 to 9. During an extensive search on different hypersaline habitats in Spain and 
Morocco focused on the screening of new exopolysacharide (EPS) -producing bacte- 
ria, several strains were isolated from saline soils and described as new species 
belonging to the genus Halomonas: Halomonas eurihalina (formerly Volcaniella 
eurihalina; Quesada et al. 1990, 1993), Halomonas maura (Bouchotroch et al. 2001), 
Halomonas ventosae (Martinez-Canovas et al. 2004b), and Halomonas anticariensis 
(Martinez-Canovas et al. 2004a). Other halophilic EPS -producing species were also 
isolated in these studies: Salipiger mucosus, that was the first moderately halophilic 
EPS-producing micro-organism belonging to the Alphaproteobacteria (Martinez- 
Canovas et al. 2004c) and Palleronia marisminoris, that also belongs to the 
Alphaproteobacteria and was isolated from a saline soil bordering a saltern on the 
Mediterranean coast near Murcia, south-east Spain (Martinez-Checa et al. 2005). 

The genus Marinobacter, with the type species Marinobacter hydrocarbono- 
clasticus, was created in 1992 to accommodate Gram- negative, moderately halo- 
philic, aerobic Gammaproteobacteria that utilize a variety of hydrocarbons as the 
sole source of carbon and energy (Gauthier et al. 1992). Currently, the genus com- 
prises 13 species, some of which being moderately halophilic bacteria isolated from 
soil samples: Marinobacter lipolyticus, that shows lipolytic activity with potential 
industrial applications (Martin et al. 2003), Marinobacter excellens (Gorshkova et 
al. 2003), Marinobacter sediminum (Romanenko et al. 2005), and the recently 
described Marinobacter koreensis (Kim et al. 2006). 

Obligately anaerobic bacteria that exist in extremely hypersaline lake ecosys- 
tems are quite numerous, because in these environments oxygen availability is low 
due to poor solubility, and organic carbon availability is high because substrate 
from primary production is not degraded by secondary consumers (plants, animals). 
The anoxic sediments of hypersaline environments are often characterized by a 
large number of halophilic anaerobic organisms belonging to the domain Bacteria. 



100 A. Ventosa et al. 

So far, the order Halanaerobiales contains 24 species, grouped into 1 1 genera and 2 
families, Halanaerobiaceae and Halobacteroidaceae (Oren et al. 1984; Rainey et al. 
1995; Oren 2000). Studies on solar salterns of the French Mediterranean coast 
(Salin-de-Giraud, Carmargue, Rhone Delta), permitted the isolation of several 
fermentative and halophilic bacteria from the sediments of hypersaline lagoons or 
from the surface of certain hypersaline ponds characterized by gypsum deposits. The 
salinities ranged from 13 to 34% NaCl (Caumette 1993; Caumette at al. 1994). 

Most fermentative bacterial isolates were assigned to the family Halobactero- 
idaceae. One of these isolates was described as a new species, Halanaerobacter 
salinarius (Moune et al. 1999), isolated from the black anoxic sediment of these 
ponds, where it co-exists with the sulfate reducers. Among the other strains isolated 
in this sampling, strain SG 3902 was phylogenetically related to the genus Orenia 
according to 16S rRNA gene sequence comparison. Whereas this genus was ini- 
tially represented by a single species, Orenia marismortui, isolated from the Dead 
Sea (Rainey et al. 1995), isolate SG 3902 showed sufficient physiological and 
genetic differences from the species O. marismortui to be considered as a new 
member of the genus Orenia, designated Orenia salinaria (Moune et al. 2000). 
Another halophilic species of the family Halobacteroidaceae is Natroniella 
acetica, an extremely haloalkaliphilic, chemo-organotrophic, homoacetogenic 
bacterium isolated from the bottom mud of the soda-depositing Lake Magadi in 
Kenya (Zhilina et al. 1996). 

The other family of the order Halanaerobiales is Halanaerobiaceae, that comprises 
one genus, Halanaerobium. This genus was described by Zeikus and coworkers in 
1983 and comprises nine species, some of which are halophilic species isolated from 
soil samples. The first species included in this genus was Haloanaerobium praevalens, 
a species with an extremely halophilic response (Zeikus et al. 1983), isolated during 
a study carried out in 1979 on the microbial ecology of anaerobic decomposition 
processes in Great Salt Lake (Utah). The objective of the study was to understand 
how organic matter is degraded in a productive hypersaline environment and what 
bacterial species actively participate in this process (Zeikus, 1983). A subsequent 
screening study of the Great Salt Lake led to the description of Halanaerobium 
alcaliphilum, an alkalitolerant species able to grow at a range of pH values from pH 
5.8 to 10, with an optimum between pH 6.7 to 7.0 (Tsai et al. 1995). 

Two other species, Halanaerobium saccharolytica subsp. senegalensis (formerly 
Haloincola saccharolytica subsp. senegalensis; Cayol et al. 1994b) and 
Halanaerobium lacusrosei (Cayol et al. 1995), were isolated from 1.5-m deep sedi- 
ment of hypersaline Lake Retba, near Dakar (Senegal); this lake is located 100m 
from the Atlantic Ocean. Finally, within this family, the genus Halothermothrix 
comprises only one halophilic species, H. orenii, to have been isolated from a soil 
habitat. This was the first described moderately halophilic (with an optimal growth 
between 5 and 17.5% NaCl) thermophile that showed a capacity to ferment carbo- 
hydrates to acetate, ethanol, H 2 , and C0 2 . This species, which was isolated from 
sediments of a hypersaline Tunisian lake (Chott El Guettar), extended the tempera- 
ture limit of halophilic anaerobic bacteria to 68 °C. The optimum temperature for 
growth of this species is 60°C (Cayol et al. 1994a). 



5 Halophilic and Halotolerant Micro-Organisms from Soils 101 

5.4 Biotechnological Applications 

A great metabolic diversity has been found in halophilic and halotolerant micro-organ- 
isms isolated from soils, and in fact many of them have biotechnological potential. 
Thus, some of them produce halophilic proteins useful in specific transformations, 
and others excrete products such as compatible solutes and biopolymers of great 
interest in different industries. In addition, these micro-organisms could be used in 
environmental bioremediation processes. The biotechnological applications of 
halophilic micro-organisms have been reviewed in detail (Ventosa et al. 1998; 
Margesin and Schinner 2001; Mellado and Ventosa 2003). In this chapter, we focus on 
interesting aspects of halophilic and halotolerant micro-organisms from saline soils. 



5. 4. 1 Extracellular Enzymes 

Industrial biocatalysis has found in the halophilic micro-organisms a source of 
enzymes with novel properties of high interest. Over the years, different enzymes 
of halotolerant and halophilic micro-organisms isolated from saline soils have been 
described and a number of new possibilities for industrial processes have emerged 
due to their overall inherent stability at high salt concentrations. These enzymes 
could be used in harsh industrial processes such as food processing, biosynthetic 
processes, and washing (Ventosa et al. 2005). 

Halophilic enzymes are active and stable at high salt concentrations, showing 
specific molecular properties that allow them to cope with osmotic stress. In gen- 
eral, these enzymes present an excess of acidic residues over basic residues and a 
low content of hydrophobic residues at their surface (Mevarech et al. 2000). The 
hydrolases able to break down various polymers constitute the group of highest 
biotechnological interest, catalyzing conversions under conditions compatible with 
industrial applications. So far, most of the characterized halophilic enzymes have 
been obtained from micro-organisms isolated from hypersaline or saline water sys- 
tems, such as lakes or salterns (Ventosa et al. 2005). We focus only on the few 
halophilic enzymes produced by micro-organisms isolated from saline soils. 

Amylases produced by several halophilic bacteria isolated from saline soils have 
been reported. Nesterenkonia halobia (formerly Micrococcus halobius) produces 
an amylase showing high dependency on salt for activity; thus, in the absence of 
high concentrations of NaCl or KC1 the enzyme loses its activity (Onishi and 
Kamekura 1972; Onishi 1972). Onishi and Hidaka (1978) reported the purification 
of two amylases from the culture filtrate of Acinetobacter sp., a moderately halo- 
philic bacterium isolated from sea-sands. The enzymes showed maximal activity in 
saline media containing 0.2-0.6 M NaCl or KC1, at pH 7.0 and 50-55°C. 

Halothermothrix orenii, a thermophilic, halophilic anaerobic bacterium isolated 
from the sediment of a Tunisian salted lake, produces two amylases, designated as 
AmyA and AmyB . The gene encoding AmyA has been cloned, characterized, and 
expressed in Escherichia coli (Mijts and Patel 2002). The recombinant enzyme 



102 A. Ventosa et al. 

presented optimal activity at 65°C in 5% NaCl and pH 7.5, although a significant 
activity was also measured in the presence of up to 25% NaCl. Li et al. (2002) have 
performed a crystallographic study of Amy A. The crystal structure of the enzyme 
reveals the lack of the acidic surface which is characteristic of halophilic proteins. 
AmyB is a 599-residue protein active and stable at 10% NaCl, the structure of 
which has also been determined by X-ray crystallography (Tan et al. 2003). 

Another extracellular amylase produced by the moderately halophilic, Gram- 
positive, spore-forming bacterium Halobacillus karajensis (Amoozegar et al. 
2003b) has been studied. This bacterium was isolated from surface saline soil of the 
Karaj region, Iran. The maximum protease activity was achieved in the presence of 
5% NaCl at pH 7.5-8.5 and 50°C (Amoozegar et al. 2003a). 

A halophilic member of the genus Bacillus, Bacillus sp. no. 21-1, produces a 
protease showing maximal activity at 5M NaCl and 0.75 M KC1 (Kamekura and 
Onishi 1974). Another extracellular protease has been characterized from a haloal- 
kaliphilic bacterium isolated from salt-enriched soil samples collected from saline 
habitats from the Veraval costal region of Gujarat, India. This bacterium has been 
identified as Bacillus pseudofirmus and the maximal enzyme production (410U/ml) 
is achieved during the early stationary phase of growth (Patel et al. 2005). 

Several moderately halophilic bacteria able to produce extracellular enzymes of 
industrial interest have been isolated in the course of a screening program per- 
formed in saline waters and soils in South Spain (Sanchez-Porro et al. 2003). A 
total of 892 strains able to produce different hydrolases were isolated: amylase (269 
strains), lipase (207 strains), protease (201 strains), DNAse (118 strains), and pul- 
lulanase (97 strains). Most of the strains showing hydrolytic activities were isolated 
from saline aquatic habitats, although surprisingly a higher number of isolates from 
saline soils produced lipolytic activity. 

The lipolytic strain SMI 9 isolated from saline soil was selected for further char- 
acterization. This bacterium has been proposed as a new species and named 
Marinobacter lipolyticus (Martin et al. 2003). The gene encoding the enzyme was 
cloned by screening an expression library in E. coli. This lipase gene, designated 
UpM, encodes a protein of 271 amino acids, with an estimated molecular mass of 
30.5 kDa. The deduced amino acid sequence of the UpM gene exhibited significant 
amino acid sequence identity with lipases belonging to the oc/p-hydrolase super- 
family (Martin et al. unpublished results). 

Several extracellular enzymes from haloarchaea have been characterized (Ventosa 
et al. 2005), however, these have been isolated from micro-organisms inhabiting 
hypersaline aquatic systems. In this sense, the future characterization of novel 
enzymes from halophilic bacteria or archaea isolated from saline soils constitutes an 
interesting research topic due to their potential biotechnological applications. 



5.4.2 Production of Compatible Solutes 

Compatible solutes are low-molecular weight organic compounds such as polyols, 
amino acids, sugars, and betaines that the halophilic and halotolerant bacteria 



5 Halophilic and Halotolerant Micro-Organisms from Soils 103 

accumulate intracellularly to achieve osmotic balance (Brown 1976). These 
compounds have industrial applications as stabilizers of enzymes, nucleic acids, 
membranes, and whole cells against stresses such as high temperature, desiccation, 
and freezing (Louis et al. 1994; Nieto and Vargas 2002). Only a few studies deal with 
the biosynthesis of compatible solutes by moderate halophiles isolated from soils. 

Ectoines constitute the predominant class of osmolytes accumulated by bacteria 
grown at high salt concentrations (Galinski and Tindall 1992). The accumulation 
of ectoine has been probed in the Gram-positive moderate halophile Marinococcus 
halophilus and the genes encoding the biosynthetic pathway of the compatible 
solute have been characterized (Louis and Galinski, 1997). Halomonas elongata 
strain KS3 is a moderately halophilic bacterium isolated from a salty soil in 
Thailand. This strain accumulates ectoine and hydroxyectoine as compatible sol- 
utes. The accumulation of these compounds is stimulated by hyperosmotic stress 
induced by salt. Ectoine production reaches approximately 120|lg mg _1 of dry cells 
at a concentration of 2.56 M NaCl and hydroxyectoine accumulates to 45|lg mg _1 
of dry cells at a concentration of 2.56 M NaCl (Ono et al. 1998). The enzymes 
involved in the biosynthesis of ectoine in H. elongata KS3 have been characterized 
(Ono et al. 1999). 

A similar ectoine biosynthetic pathway has been found in Chromohalobacter 
salexigens, a moderately halophilic bacterium that has been used as a model 
micro-organism for the study of osmoadaptation mechanisms in moderate halo- 
philes (Canovas et al. 1997, 1998). The use of ectoine as a stabilizer of different 
enzymes (amylases, lipases, cellulases, proteases) has been patented (Toyoda et al. 
1997). Moreover, ectoine and ectoine derivatives have been patented as moisturiz- 
ers in cosmetics (Motitschke et al. 2001). On the other hand, Nakayama et al. 
(2000) expressed the ectoine biosynthetic genes in cultured tobacco, obtaining an 
increase in the tolerance of the transgenic plant to hyperosmotic stress. This is an 
interesting result, especially considering that in arid and semi-arid lands environ- 
mental stress limits plant growth and productivity. 

Halobacillus halophilus (formerly Sporosarcina halophila) is a moderately 
halophilic bacterium isolated from salt marsh soils of the North Sea coast of 
Germany (Claus et al. 1983). This bacterium produces glutamate and glutamine as 
its main compatible solutes and thus it could be used for the industrial production 
of these compounds. Recently, Saum et al. (2006) described the routes for the bio- 
synthesis of these solutes and characterized the genes involved in their synthesis. 



5.4.3 Production of Exopolysaccharides 

Several moderately halophilic bacteria isolated from saline soils are able to 
produce exopolysaccharides which can be secreted in the extracellular medium in 
the form of amorphous slime. The exopolysaccharides have biotechnological 
interest due to both viscosifying and emulsifying properties, which allow them to 
be used in medicine, pharmacy, foodstuffs, cosmetics, and the petroleum industry 
(Quesada et al. 2004). 



104 A. Ventosa et al. 

The genus Halomonas groups several species able to produce exopoly saccharides. 
Halomonas eurihalina (formerly Volcaniella eurihalina) has been described 
as a producer of a exopolysaccharide with the ability to increase the viscosity of 
solutions at low pH values (Martinez-Checa et al. 2002). Halomonas maura, 
isolated from soil samples collected from a saltern at Asilah (Morocco), produces 
an exopolysaccharide designated Mauran that has been studied in detail. This 
polymer is highly viscous and shows thixotropic and pseudoplastic behaviour pre- 
senting interesting properties for biotechnology (Arias et al. 2003). The genes 
involved in the biosynthesis of this exopolysaccharide have been cloned and the 
maximum induction is reached during the stationary phase in the presence of 5% 
marine salts (Arco et al. 2005). Halomonas ventosae, isolated from saline soils in 
Jaen (Spain), and Halomonas anticariensis, isolated from soil samples taken from 
Fuente de Piedra, a saline wetland in Malaga, South Spain, produce exopolysac- 
charides showing emulsifying activity on several hydrophobic substrates (Mata et 
al. 2006). In addition to the genus Halomonas, other moderately halophilic bacteria 
isolated from hypersaline soils, such as Salipiger mucescens (Martinez-Canovas et 
al. 2004c) and Palleronia marisminoris (Martinez-Checa et al. 2005), have also 
been described as exopolysaccharide producers; however, the features of these 
exopolysaccharides have not been investigated in detail. 



5. 4. 4 Production of Carotenoids 

Most haloarchaea inhabiting hypersaline habitats synthesize carotenoids that 
contribute to the red color presented by many saltern crystallizer ponds and 
hypersaline lakes (Kushwaha et al. 1974; Oren 2002). Industrial applications of 
carotenoids include their use as nutrient supplements, as food colorants, in the 
pharmaceutical industry, or in animal feeds. In this sense, Asker and Ohta (1999) 
used soil samples from a salt farm as a source for the isolation of carotenoid- 
producing micro-organisms; they isolated 31 red haloarchaea that might represent 
valuable sources of carotenoids. 



5.4.5 Biodegradation of Toxic Compounds 

Industrial processes such as the production of pesticides, herbicides, and pharma- 
ceutical products, in addition to paper mills and petrochemical industries generate 
wastewaters containing toxic compounds and with different levels of salinities. 
According to environmental regulations, these residual waters need to be treated 
prior to their release in the environment. Some of the treatments conventionally 
used combine physicochemical and biological methods. In the biological treatment, 
the micro-organisms conventionally used show only poor degradative efficacy due 
to the highly saline conditions. The degradation of aromatic compounds in saline 
conditions using the potential of halophilic bacteria is of great interest and offers 



5 Halophilic and Halotolerant Micro-Organisms from Soils 105 

the promise of innovative research in saline soil microbiology and of new treatment 
strategies for saline effluents generated by different industrial activities. 

The metabolism of aromatic compounds in halophilic archaea was demonstrated 
for the first time in Haloferax sp. D1227, isolated from soil contaminated with highly 
saline oil brine (Fu and Oriel 1998, 1999). Several studies have dealt with the isolation, 
from saline contaminated soils, of moderate halophiles with the ability to degrade 
different organic compounds. The moderately halophilic bacterium Arhodomonas 
aquaeoli, isolated from subterranean brines, has the ability to aerobically degrade oil 
(Adkins et al. 1993). Recently, a study focused on the characterization of an active 
and acclimated bacterial population able to degrade aromatic compounds in saline 
habitats of South Spain has been carried out (Garcia et al. 2005b). Two novel moder- 
ately halophilic bacteria isolated from saline soils, designated as Halomonas 
organivorans and Thalassobacillus devorans have been described. H. organivorans 
is a Gram-negative bacterium with the ability to use a broad variety of organic 
compounds (benzoic acid, p-hydroxybenzoic acid, cinnamic acid, salicylic acid, 
phenylacetic acid, phenylpropionic acid, phenol, p-coumaric acid, ferulic acid, and 
^-aminosalicylic acid; Garcia et al. 2004). Thalassobacillus devorans is a Gram-positive 
bacterium isolated by phenol enrichment of samples collected in contaminated 
hypersaline soils (Garcia et al. 2005a). Both novel species have the potential to be 
used for the biodegradation of contaminated saline habitats. 

Organophosphonates constitute toxic contaminants due mainly to the stable cova- 
lent carbon to phosphorus (C-P) bond. The contamination of environments with these 
recalcitrant compounds constitutes a serious environmental problem. Hayes et al. 
(2000) isolated a halophilic strain from soil beneath a road gritting salt pile with the 
ability to utilize phosphonoacetate, 2-aminoethyl-, 3-aminopropyl-, 4-aminobutyl-, 
methyl- and ethyl-phosphonates as phosphorus sources for growth. The novel isolate 
was shown to be probably Chromohalobacter marismortui or Chromohalobacter 
(Pseudomonas) beijerinckii on the basis of 16S rRNA analysis. 

Recently, Kleinsteuber et al. (2006) have performed microbiological examina- 
tion of an exploited oil field with high soil salinity near Comodoro Rivadaria in 
Patagonia (Argentina). They characterized a community of halophilic bacteria with 
a capacity for diesel fuel degradation and identified the most active species by high- 
resolution cell sorting and analysis of the 16S rRNA gene. 

On the other hand, halophilic bacteria tolerant to heavy metals could be used as 
bioassay indicator organisms in saline-polluted environments. Several halotolerant 
and halophilic bacteria isolated from hypersaline soils tolerate high concentrations 
of different metals, such as Co, Ni, Cd, or Cr (Nieto et al. 1989; Rios et al. 1998). 



5.5 Conclusions 

Saline and hypersaline soils are increasingly abundant as a consequence of the 
irrigation and desertization processes. The study of the microbiological activities in 
these habitats yields results of great interest. Halophilic and halotolerant micro-organisms 
are the most important microbial population of these soils. The microbial diversity 



106 A. Ventosa et al. 

of saline soils is more similar to that of nonsaline soils than to the microbiota found 
in hypersaline water systems. Many studies have been focused on the isolation and 
characterization of halophilic bacteria and archaea from saline or hypersaline soils. 
Biotechnological applications of these micro-organisms have been or are under 
investigation. However, more extensive studies on the ecology, biochemistry, and 
physiology of organisms belonging to the different microbial groups present in 
these soils are urgently required. 

Acknowledgements The authors' research was supported by grants from the Quality of Life and 
Management of Living Resources Programme of the European Commission (QLK3-CT-2002- 
01972), Spanish Ministerio de Education y Ciencia (projects BIO2006-06927 and CTM2006- 
03310) and Junta de Andalucia. 



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56:839-844 



Chapter 6 

Atacama Desert Soil Microbiology 



Benito Gomez-Silva(S), Fred A. Rainey, Kimberley A. Warren-Rhodes, 
Christopher P. McKay, and Rafael Navarro-Gonzalez 



6.1 Introduction 

The Atacama Desert is an ancient temperate desert (mean annual temperature of 
14-16°C) that extends across 1,000 km from 30°S to 20°S along the Pacific coast of 
South America (McKay et al. 2003; Fig. 6.1). As discussed by Rundel et al. (1991) and 
Miller (1976) the desert owes its extreme aridity to the climatic regime dominated by a 
constant temperature inversion due to the cool north-flowing Humboldt Current and the 
presence of the strong Pacific anticyclone. The position of the Pacific anticyclone is gen- 
erally stable with a small shift of a few degrees south in the summer (Trewartha 1961). 
Geological and soil mineralogical evidence suggests that extreme arid conditions have 
persisted in the Southern Atacama for 10-15 million years (Myrs; Ericksen 1983; 
Houston and Hartley 2003; Clarke 2006) making it one of the oldest deserts on Earth. 

The driest parts of the Atacama Desert are located between approximately 22°S 
to 26 °S in the broad valley formed by the coastal range and the medial range (the 
Cordillera de Domeyko) (Fig. 6.2). One of the most striking and unusual features 
of the Atacama Desert is the presence of large nitrate deposits. Early in the last 
century, nitrate mining operations were conducted in this area (Woodcock and Hill 
1901), but, currently, there are no permanent human settlements in this hyperarid 
region. Although nitrate deposits are found in many deserts, significant accumula- 
tions are found only in the Atacama. Current understanding suggests that the nitrate 
is of atmospheric (lightning) origin based on stable isotope evidence (as suggested 
by Bohlke et al. (1997)) and that lightning-related nitrate production is not 
unusually intense over the Atacama. What is unusual is that there are no removal 
mechanisms due to the lack of water activity and resultant lack of microbial deni- 
trification. Over the history of the Atacama (10-15 Myr or more) the accumulation 
has resulted in large concentrated deposits. 

Within the driest part of the Atacama there exists a region with "Mars-like" soils 
(Navarro-Gonzalez et al. 2003). There are three characteristics that make these soils 



Benito Gomez-Silva 

Institute* del Desierto y Unidad de Bioqufmica, Facultad Ciencias de la Salud, Universidad de 

Antofagasta, Casilla 170, Antofagasta, Chile 

e-mail: bgomez@uantof.cl 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 1 17 

© Springer- Verlag Berlin Heidelberg 2008 



118 



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Fig. 6.1 Map of the Atacama Desert in Northern Chile showing the location of the hyperarid core 
near Yungay (24°S). (Adapted from McKay et al. 2003) 



6 Atacama Desert Soil Microbiology 119 

LicHncAtxjr Volcano Junquos Volcano Ml Zap 
S916iTi 5704 m 
Vcute Mackonna Htns. 
31 14 m 

Mejitones Peninsula 




Uttofai Platform Coastal Htns. Atacama Oesafl Domeyto Mtns. Andes Dftpf«$*on Andes Mountains 

Fig. 6.2 Cross-section of the Atacama Desert region near Antofagasta, Chile. (Adapted from 
Guias Turistel, Ediciones Turiscom) 



Mars-like: first, there are very low levels of organic material and the organics that 
are present are refractory. They do not decompose at the temperatures reached by the 
Viking GCMS (500°C). Second, there are very low levels of soil bacteria, and, in 
some locations, these are undetectable either by culture or DNA amplification 
(Navarro-Gonzalez et al 2003), or by Limulus Amebocyte Lysate, a sensitive and 
specific assay to screen for the presence of endotoxin (lipopolysaccharide) from 
Gram-negative bacteria (M. Turnbull, personal communication). Third, the soil 
contains an oxidizing agent with the ability to oxidize at equal rates l- and d- amino 
acids, as well as l- and d- sugars. 

Soils to the south of the arid core region do not show these characteristics. 
We think these Mars-like characteristics result from the extreme aridity of the core 
region of the Atacama. The entire Atacama is arid and receives very little rain. 
However, many locations in the desert receive marine fog providing sufficient mois- 
ture for hypolithic algae, lichens, and even cacti (Rundel et al. 1991; Warren-Rhodes 
et al. 2006). However, in the region south of Antofagasta the coastal range blocks the 
marine fog. The crest-line of the coastal range averages 3,000m for about 100km 
south of Antofagasta. The region that is in the "fog shadow" of this high coastal crest- 
line is the region that contains the Mars-like soils. Rech et al. (2003) have used sulfur 
isotopes to trace out the areas affected by marine input. Their results are consistent 
with the coastal range blocking the marine fog in the region of Mars-like soils. Thus 
the Atacama Desert is in the rain shadow of the Andes and the cold Humboldt 
Current. The Mars-like soils are found in those regions that are even drier yet because 
they are also in the fog shadow of the coastal range. 

In this chapter we discuss the microbiology of the extreme arid core of the 
Atacama Desert and compare it to less arid regions of that desert. We then discuss 
how the arid core region provides a test bed for the future search for life on Mars. 



6.2 Atacama Soil Organics 

Soil organics are a mixture of recognizable biological material whose components 
have been altered to the degree that it no longer retains its original structural or 
chemical organization (Oades 1989). At any given time the amount of soil organics 



120 B. Gomez-Silva et al. 

reflects the long-term balance between input and loss rates (Olson 1963). The input 
of soil organics increases with mean annual precipitation and temperature, whereas 
their residence time decreases with mean annual precipitation and mean annual 
temperature (Lieth 1973; Amundson 2001). Soil organics are ultimately degraded 
to carbon dioxide through microbial respiration and abiotic processes (Bunt and 
Rovira 1954; Parsons et al. 2004). 

Organics in surface samples of Atacama Desert soils have been studied in detail 
using dry analytical methods, namely pyrolysis-gas chromatography-mass spectrometry 
(pyr-GC-MS) at atmospheric pressure in an inert atmosphere by Navarro-Gonzalez 
et al. (2003). Samples exposed to flash heating at 500°C in a He atmosphere revealed 
that the most arid zone of the Atacama, the Yungay area (~24°S) is depleted of organic 
molecules. At a higher temperature pyrolysis (750°C), only formic acid and benzene 
are detectable. In contrast, samples from less arid sites (28°S) release a complex 
mixture of organic compounds upon pyrolysis at 750°C (formic acid, propenenitrile, 
1,2-butadiene, 2-butene, 1,3-pentadiene, 2-methylfuran, benzene, methylbenzene, 
benzenenitrile, ethylbenzene, 1 ,2-dimethylbenzene, and styrene). A comparative 
analysis of this thermal treatment with proteins, peptides, and free amino acids yielded 
a series of carboxylic acids, saturated nitriles, and saturated, unsaturated, and aromatic 
hydrocarbons; carbohydrates were degraded to a series of aliphatic aldehydes, ketones, 
carboxylic acids, aromatic compounds, and furan derivatives; fatty acids were pyrolyzed 
to alkanes, alkenes, aromatic compounds, and short chain carboxylic acids; porphyrins 
were degraded to pyrroles; and nucleic acid bases released unsaturated nitriles and 
substituted furans. Pyrolysis of Atacama Desert bacterial isolates (e.g., strain AT01-3, 
isolated from soil samples at S 24° 4' 9.6", W 69° 51' 58.8", Navarro-Gonzalez et al. 
2003) released a mixture of all the above classes of organic compounds. 

From these results, it was concluded that Atacama sites at 28°S contain bacteria 
and/or all of the major classes of biomolecules at levels within the detection limits 
of the pyr-GC-MS protocol applied (Navarro-Gonzalez et al. 2003). The two 
characteristic compounds released by pyrolysis at 750°C in a He atmosphere by all 
samples of the Atacama Desert along a precipitation gradient from 24° S to 28 °S 
are formic acid, a highly oxidized organic compound, and benzene, a thermally 
stable aromatic compound. These two compounds are typically released by the 
thermal treatment of monocarboxylic acids, polycarboxylic acids, carbohydrates, 
polysaccharides, amino acids, and proteins. 

Formic acid is present at concentrations of ~50|lg/g in the Yungay area (~24°S), 
then decreases by an order of magnitude at 26°S, and increases again in the less arid 
zone. In contrast, benzene is present at trace levels (~l-2|ig/g) at ~24°S with its 
concentration increasing to ~80|lg/g in the less arid zone (~28°S). The ratio 
between formic acid and benzene reaches its highest value (> 15 units) in the 
Yungay area, and then sharply drops to <0.3 from 25°S to 28°S. A high formic 
acid/benzene ratio indicates that the organic matter present in the region is oxidized, 
and possibly composed of refractory organics such as aliphatic and aromatic mono- 
and polycarboxylic acids (Navarro-Gonzalez et al. 2003). 

The organics present in the Yungay soils are dominated by carboxylic acids and 
polycyclic aromatic hydrocarbons as determined by nuclear magnetic resonance 



6 Atacama Desert Soil Microbiology 121 

(NMR) and infrared spectroscopy (IR) analysis. The 8 13 C values of soil organics 
varied from -26 A%o at 24°S to -28.9% at 28°S (Navarro-Gonzalez et al. 2006), a 
typical range for organic matter produced by C 3 photosynthesis (O'Leary 1998). 
Similarly the C/N ratio for these samples varied from 8.2 at 24°S to 16.7 at 28 °S 
(Navarro-Gonzalez et al. 2006), a typical range for soil organic matter (Batjes 
1996). Surprisingly, the levels of total organics determined by wet analytical 
methods (titration by oxidation with permanganate) in the Atacama Desert soil are 
much higher than the levels derived by pyr-GC-MS (Navarro-Gonzalez et al. 2006). 
The concentration of organics, as determined by permanganate oxidation, is 
-0.04 mg of carbon per gram of soil in the Yungay area, and increases with precipi- 
tation to -0.70 mg of carbon per gram of soil at -28 °S. Considering that 1 |lg of 
benzene is equivalent to 0.92 |ig of carbon, it appears that the organic matter content 
is underestimated by pyr-GC-MS by a factor varying from 20 to 40 in the Yungay 
region to about 10 at ~28°S. These results show an important limitation of the 
pyr-GC-MS technique for detection of organic material. When organics are present 
as low-level refractory substances, the temperatures reached by the pyrolysis 
method (500-750°C) are inadequate to release all the organics for detection. 

Free carboxylic and amino acids present in the Atacama soil at a sample site 
situated at approximately ~27°S have been analyzed by solvent extraction (1:1 
mixture isopropanol and water assisted by ultrasonic treatment), followed by 
chemical derivatization and gas chromatography-mass spectrometry (Buch et al. 
2006). The following compounds were detected at |ig/g levels: benzoic acid, 0.33; 
nonanoic acid, 0.32; 3-methyl benzoic acid, 0.01; 2-butanoic acid, 0.01; hexanoic 
acid, 0.52; glycine, 0.02; alanine, 0.09; sarcosine, 0.02; valine, 0.10; and norvaline, 
<0.01 (Buch et al. 2006). Total amino acids (free and chemically bound) in the 
Atacama soil precipitation gradient have been analyzed by acid hydrolysis followed 
by capillary electrophoresis and/or HPLC. The level of glycine varies from 0.03 |ig/g, 
at 24.1°S to 0.02 ng/g at 25.8°S, and 0.32 ^g/g at 28.2°S (Skelley et al. 2005). 

Although amino acids are the single most abundant compounds in bacteria (e.g., 
E. coli) cells (Neidhardt et al.1990), it is known that most (>98%) of the amino 
acids originally present in bacterial cells do not undergo sublimation and are 
destroyed during pyrolysis under vacuum (Glavin et al. 2001). Even though nucleic 
acid bases are less abundant than amino acids, accounting for only 24% of the dry 
E. coli cell weight (Neidhardt et al. 1990), purines and pyrimidines are much more 
resistant to thermal degradation than protein-bound amino acids and readily 
sublimate directly from DNA and RNA when cells are heated (Glavin et al. 2002). 
Trace levels of nucleobases (5 to 76 ng/g) were identified in the sublimed extract of 
the Atacama Desert in the Flat Top Hill site (approximately 160 km south of the 
Yungay region). Based on the amount of adenine sublimed (5.4 ng/g), Glavin at al. 
(2004) estimated a total bacterial density of 4.4 x 10 6 cells per gram. 

Because of the lack of water availability in the soil, there is virtually no primary 
productivity in this zone. Hypolithic cyanobacteria colonize only about 28% of 
translucent stones in the less arid zone of the Atacama Desert, around ~27°S, but 
these bacteria are extremely rare in the core region of the Atacama, colonizing less 
than 0.1% of the translucent rocks in the Yungay region (~24°S; Warren-Rhodes 



122 B. Gomez-Silva et al. 

et al. 2006). Culturable heterotrophic bacteria are present in the less arid region of 
the Atacama Desert at levels of 10 7 colony-forming units per gram of soil. However, 
in the core region the levels of culturable heterotrophic bacteria are extremely low, 
corresponding to between < 10 2 to 10 4 colony-forming units per gram of soil from 
24° to 25°S (Navarro- Gonzalez et al. 2003). Therefore, microbial degradation of 
soil organic matter (SOM) in this region is extremely restricted, implying that the 
degradation of SOM must be controlled mainly by abiotic processes. 

Photodegradation is thought to be the dominant abiotic process controlling the 
decomposition of SOM (Austin and Vivanco 2006). The ultraviolet flux along 
the transect from 24° to 25 °S is not significantly different because the region is only 
1 km above sea level, and the zone has clear blue skies all year around (McKay et al. 
2003). Therefore, the hyperarid conditions in the Yungay area must inhibit microbial 
survival and the net result is that photochemical processes dominate. This is the first 
known region of the planet in which the degradation of SOM is mainly controlled 
by abiotic processes. In another extreme hyperarid area, the Dry Valleys in the 
Antarctica, it has been shown that the abiotic processes may be as important as the 
biological processes in the degradation of SOM to carbon dioxide (Parsons et al. 
2004). These soils have significantly higher levels of organics, from 0.24 to 0.34 mg 
of carbon per gram of soil (Parsons et al. 2004), as compared to -0.04 mg of carbon 
per gram of soil in the Yungay area (see above, this section). 



6.3 Isolation and Detection of Heterotrophic Bacteria 

There is currently a limited amount of published information on the levels of heter- 
otrophic bacteria and their identity from the Atacama Desert. Studies to date have 
estimated that desert soil populations ranged from to 10 7 colony-forming units per 
gram (CFU/g). This wide range of values is not surprising considering the extent of 
the desert, the various levels of precipitation, as well as the chemical compositions 
and elevations of various sample sites. Apparent differences in population size 
might also correspond to differences in efficiency of the recovery methods used. 
Population size estimates were obtained using both culture-dependent (Cameron 
1969a,b; Navarro- Gonzalez et al. 2003; Maier et al. 2004; Lester et al. 2007) and 
culture-independent approaches (Glavin et al. 2004; Drees et al. 2006). The culture- 
dependent approaches which involve the growth of viable cells on artificial culture 
media are for the most part in agreement and conclude that the bacterial numbers 
contained within the soils from the most arid region of the Atacama are extremely 
low and in most cases close to or below the detection limit of the methods applied. 
The population density values reported from culture-dependent studies of Atacama 
soil samples are also very low when compared to results from similar studies of 
other arid or nonarid soils (Navarro-Gonzalez et al. 2003). 

In the recent literature, the first estimation of bacterial population density comes 
from the study of Navarro-Gonzalez et al. (2003) in which samples along a precipi- 
tation gradient from north to south were studied. The CFU/g values from that study 



6 Atacama Desert Soil Microbiology 123 

ranged from < 10 3 (in the hyperarid core region at 24° S) to > 10 5 (in the most south- 
ern sample site at 28 °S). The bacteria isolated in that study were mainly species of 
the Actinobacteria and Firmicutes (or Gram-positive bacteria with a low G+C 
content), with only a small number of Proteobacteria being recovered. This study 
showed a decrease in the number of cultivable heterotrophic bacteria in surface 
soils along the south to north precipitation gradient of the Atacama Desert. Together 
with this decrease in numbers, a corresponding decrease in diversity based on 
taxonomic assignment was observed. 

In the study of Lester et al. (2007), a total of 20 isolates was identified by 16S 
rRNA gene sequence comparisons and found to be Alphaproteobacteria (12 strains), 
Betaproteobacteria (1 strain), and Bacillus species (7 strains). Of the seven Bacillus 
strains, five were identified as B. pumilus strains, and the Alphaproteobacteria were 
related to the genera Sphingomonas, Asticcacaulis, Mesorhizobium, Bradyrhizobium, 
and Afipia. These isolates came from three samples at a specific site and were isolated 
on Trypticase Soy Agar and R2A agar (Difco Catalog). In the same study (Lester et 
al. 2007), culture-independent methods yielded a small number of 16S rRNA gene 
sequences from 12 denaturing gradient gel electrophoresis (DGGE) bands. These 
were shown to belong to the Gemmatimonadetes, Actinobacteria, Planctomycetes, 
Thermomicrobia, and Proteobacteria. 

Although a number of these studies have been carried out in the most arid core of 
the desert, these have been conducted at sites distant from one another and bacteria 
were recovered using a variety of different culture media, making direct comparisons 
difficult. In addition, the number of samples studied at each site has been limited. 

To obtain results that would be more amenable to comparative analyses, we 
performed extensive sampling within the hyperarid core region of the desert close to 
Yungay. Within the hyperarid core region of the desert, the number of culturable 
heterotrophic bacteria in surface soil samples is extremely low. No heterotrophic 
bacteria could be cultured from two-thirds of the samples from this area that were 
used to inoculate at least one culture medium. The CFU/g values determined within 
this core region range from to 10 5 per gram of soil. The sample containing 10 5 
CFU/g is an outlier from a region in which the majority of the samples have CFU/g 
in the range to < 10 3 . The organisms cultured from these surface samples collected 
within the core region are limited in diversity and the majority belongs to the class 
Actinobacteria and more specifically to a particular group within this class, the family 
Geodermatophilaceae. The remaining organisms that were recovered belonged 
to species of the genera Sphingomonas, Bacillus, Arthrobacter, Brevibacillus, Kocuria, 
Cellulomonas, and Hymenobacter. A considerable degree of spatial heterogeneity in 
distribution of the CFU/g values is observed among the surface samples, which can- 
not be easily explained and is not highly correlated to soil chemistry. 

The same situation is found in the subsurface samples (Navarro-Gonzalez et al. 
2004). Indeed, soil pits constructed within the hyperarid core region of the Atacama 
Desert show a pattern of spatial heterogeneity similar to the surface samples. The 
CFU/g values range from to 10 3 in all layers of the soil pits sampled. No pattern 
is discernible in the spatial distribution of the bacteria isolated from the soil pits, 
with the majority of layers containing no culturable bacteria. This is in stark 



124 B. Gomez-Silva et al. 

contrast to a soil pit constructed and studied at a more southern point along the 
precipitation gradient, where CFU/g values in the range 10 3 to 10 6 are detected in 
all layers. The diversity of the bacteria isolated from the subsurface samples was 
similar to that recovered from the surface samples, except that no Proteobacteria 
were recovered. The examination of samples from the hyperarid core region con- 
sistently demonstrates that the soils of this region have extremely low numbers of 
culturable heterotrophic bacteria and that the diversity of these organisms is limited 
when compared to microbial populations from other desert soils. 

Many unanswered questions remain regarding the reasons for these low numbers 
and the limited diversity within the Atacama Desert soil microbial populations. 
Lester et al. (2007) conclude that the bacteria isolated from the single site that they 
examined were recently transported into the desert. The subsurface data would 
point against this conclusion because there are viable bacterial strains at one meter 
depth that have not been introduced to the environment recently. Future studies of 
organisms recovered from the hyperarid core region should concentrate on their 
desiccation resistance and survival under low nutrient conditions. 



6.4 Cyanobacteria-Dominated Microbial Consortia 

Liquid water is a paramount condition for life on our planet. Long-term mean 
annual rainfall in the Atacama Desert is only a few millimeters in its driest core 
(24°-25°S, 69-70°W) and increases with latitude (McKay et al. 2003). At this 
biotic extreme environment, desert pavement (surface soils mantled by gravels) is 
colonized by hypolithic or endolithic bacterial consortia forming biofilms on or 
within rock substrates such as quartz (Warren-Rhodes et al. 2006), halite (Wierzchos 
et al. 2006), and gypsum (J. Wierzchos and B. Gomez-Silva, unpublished results), 
as has been reported in other hot and cold deserts (Friedmann 1982; Schlesinger 
et al. 2003). The cyanobacterium Chroococcidiopsis sp. is the ubiquitous primary 
producer of these communities inhabiting porous and translucent stones which 
retain sufficient moisture and filter excessive solar radiation. 

Abundance of colonized stones correlates positively with the increase in the 
latitudinal rainfall gradient, as does microbial community diversity estimated on 
the basis of the recovery of 16S rRNA gene-defined genotypes (Warren-Rhodes et al. 
2006). At wetter sites, microbial consortia contain genotypes belonging to cyano- 
bacteria {Chroococcidiopsis sp. and Nostoc sp.), Alpha- and Gammaproteobacteria, 
Acidobacteriales, and Phormidium. 

Based on radiocarbon data from hypolithic biofilms and soils, microbial activity 
shows a sharp decline from wetter sites (27°S, 70°W) to the driest site at Yungay 
(24°S, 70°W), with steady-state residence times of one year and over 3,000 years, 
respectively (Warren-Rhodes et al. 2006; Fig. 6.3). At Yungay, a site with Mars-like 
soils (Navarro- Gonzalez et al. 2003), cyanobacterial colonization of stones may be 
at least 12,000 years old, comparable to Antarctic cryptoendolithic consortia 
(Bonani et al. 1988). Across the latitudinal precipitation gradient, total organic 



6 Atacama Desert Soil Microbiology 



125 



carbon of hypolithic soils is five to fifteenfold greater than organic carbon of non- 
hypolithic surface soils (Warren-Rhodes et al. 2006). Radiocarbon values also indi- 
cate that organic carbon cycling is four times greater in the hypolithic community 
of the Atacama hyperarid core than in surrounding soils, whereas at a wetter site 
(27°S, 70°W) mean turnover times were indistinguishable. These facts indicate that 
colonized stones in the Atacama are islands of fertility (Schlesinger at el. 1990) 
whose existence and spatial location is a consequence of nonuniform liquid water 
distribution. 

The spatial distribution of hypolithic communities in the Atacama is generally 
nonrandom. Transect data and spatial studies at several sites revealed that cyanobacterial 
spatial pattern is clumped, or aggregated and distinct from the background quartz 
stone pattern (Warren-Rhodes et al. 2007a). These patterns are similar to 'island- 
patch' distributions shown elsewhere for both desert vegetation and cyanobacterial 
soil crusts (Ludwig et al. 2005; Belnap et al. 2005). A detailed examination of one such 
patch in the hyperarid core (25 °S, 69°W) revealed that hypolithic communities tend to 
preferentially colonize large quartz stones (typically larger than the mean quartz stone 
size), and this may be an ecological response to extremely low water availability, 
with large stones collecting and retaining more scarce water and thus contributing 



L. 

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Fig. 6.3 The hypolithic cyanobacterial community across the moisture gradient in the Atacama 
Desert. The hyperarid core of the Atacama Desert is at 24°S. As shown in the dashed thick line, 
rainfall increases markedly south of 25 °S. Observed along this gradient were: a decrease in 
steady-state residence times of organic carbon (solid gray line, filled grey circles); an increase in 
fraction of suitable stones that are colonized by hypolithic cyanobacteria (solid black line, open 
circles); an increase in the total number of distinct sequences recovered from hypolithic communi- 
ties (dashed thin grey line, open diamonds); and an increase in total number of cyanobacterial 
sequences recovered (dashed thin black line, filled black triangles). (Adapted from Warren- 
Rhodes et al. 2006) 



126 B. Gomez-Silva et al. 

to the survival of the hypolithic or endolithic community (Warren-Rhodes et al. 
2007a). These organisms also tend to colonize particular stone aspects and certain 
locations within the environment that favor maximum moisture availability, with 
the heterogeneity in hypolithic community spatial patterns linked to nonrandom 
water distribution patterns and the factors that determine such patterns. As an example, 
many hypolithic patches in the core are located either within the landscape's extreme 
topographic lows, where moisture from rainfall converges, or in topographic highs, 
where fog moisture concentration is greatest, again suggesting that fog may play 
a role in the survival of hypolithic communities in the Atacama. 

McKay et al. (2003) have reported high nighttime relative humidity and dew in 
the central Atacama, a relatively frequent event that depends on the movement of 
moist air masses or dense fogs called camanchaca from the Pacific that reach the 
Yungay area mostly at night hours. Warren-Rhodes et al. (2006) recorded the pres- 
ence of liquid water from fog on the sides of colonized quartz stones at several sites 
along the north-south transect centered at 70° W. At these sites, a major proportion 
(60-95%) of hypolithic communities are located on stone sides, in contrast to other 
deserts such as the Mojave and Negev where hypoliths under stones are the pre- 
dominant colonization form. These ecological variations may indicate exploitation 
of more frequent fog versus rainfall moisture sources by hypolithic communities in 
the Atacama, or may be a function of other possible factors, such as rock thickness 
and orientation in relation to light availability. 

Although fog and dew are a more reliable and frequent source of moisture than 
rainfall in the Atacama' s hyperarid core, these phenomena did not contribute as 
significantly to measurable moisture on subsurface soils under stones as rare rain 
events did. Taken together, liquid water from all sources in this region is still 
exceedingly low. At Yungay, fog and rain provide liquid water to hypolithic 
cyanobacterial communities during an average of 400 h per year but water is only 
available for photosynthesis during 75 h per year (McKay et al. 2003, Warren-Rhodes 
et al. 2006). This results in an extreme environment with the lowest percentage of 
quartz stones colonized by cyanobacterial communities measured to date in the 
world's deserts (Warren-Rhodes et al. 2007b). 

Another ecological niche exploited by cyanobacterial communities may afford 
greater water availability in the Atacama 's extremely dry core. Recently, 
Wierzchos et al. (2006) have reported endolithic colonization of halite rocks at 
the Yungay area of the Atacama. These halite nonmarine evaporites are bottom- 
grown crusts shaped by aeolian action and long-term dissolution by rain and fog 
events. Using various electron microscopy techniques and fluorescent reagents, 
aggregates of Chroococcidiopsis and associated rod-shaped heterotrophic bacte- 
ria were found to be adhered to the crystal surfaces within the porous halite rocks. 
Endoevaporitic colonization of this crystalline and porous salt crust microhabitat 
may rely on the relative abundance of fog and dew events as a source of moisture. 
Deliquescence and capillarity are two processes probably involved in water 
absorption and retention. Our preliminary evidence indicates that endoevaporitic 
colonization is more broadly present in the Atacama Desert than previously envi- 
sioned. Long-term meteorological monitoring, as well as culturing and molecular 



6 Atacama Desert Soil Microbiology 127 

approaches for the study of microbial communities are presently being carried 
out to obtain a more complete picture of this new Atacama niche. 

The aridity gradient in the Atacama Desert parallels the decline in the number 
of hypolithic communities, diversity, and activity, leading from soils with a relative 
abundance of microbial life (27°S) to Mars-like soils (24°S) where life is at a 
critical biological threshold. Compared with the Antarctic desert where life is 
temperature-limited (Friedmann 1982), liquid water input in the Atacama is the key 
environmental factor that controls microbial ecology. In this warm desert environ- 
ment, temperature, habitat availability, soil pH, soil toxicity, and carbon inflow 
have a minor or null impact on photoautotrophic life. 

Contrasting with the scarcity of liquid water found along the aridity gradient and 
particularly in the hyperarid Atacama core (24°-25°S), coastal Atacama hillslopes 
are moisture-rich ecological environments owing to their function as topographic 
barriers to cloudbanks and marine air moving eastwards from the Pacific Ocean. 
Maximum daily air relative humidity in these ecosystems is greater than 80% 
during most of the year, a condition that is maintained from late afternoon to early 
morning hours (Caceres et al. 2004). Water condensation from fog and heavy dew 
provides regular moisture to subsurface soils in these environments, and unique 
coastal fog oases, or lomas, have developed, although their existence is generally 
limited to particularly fog-prone areas (Rundel et al. 1991; Munoz-Schick et al. 
2001; Thompson et al. 2003). 

Apart from coastal lomas, the remaining and larger portion of the Atacama's arid 
coastal desert, where mean annual rainfall is often <5mm/yr (18-25°S), seems devoid 
of life. However, as mentioned above (this section), hypolithic communities are also 
present here. Recent study of these coastal hypolithic communities shows that 
cultivable members of these bacterial consortia include Bacillus sp., Streptomyces sp. 
and the ubiquitous cyanobacterial primary producer Chroococcidiopsis sp. (Gomez- 
Silva et al. unpublished results). We have confirmed that accompanying Bacillus 
species biosynthesize exopoly saccharides, the composition, structure, and rheological 
properties of which are currently being assessed in order to understand their role in 
the desiccation tolerance of these micro-organisms. 

Atacama cyanobacteria- supported microbial consortia spend long periods of time 
under high solar insolation within extremely dry habitats which render cells metaboli- 
cally inactive and at high risk of undergoing macromolecular damage (Dose et al. 
2001). For example, exposure of dry B. subtilis spores to Atacama Desert's full 
sunlight at solar fluences equivalent to 16 and 300 kJ m~ 2 (at 280-320 nm) caused 
UV- B cell inactivation and 63% and 100% loss in cell viability respectively, due to 
DNA double-strand breaks (Dose et al. 2001). During rewetting periods, endolithic 
and hypolithic communities must promptly resume metabolism to sustain damage 
repair, growth, and cell division. Friedmann et al. (1993) have suggested that, in 
rapidly fluctuating environments such as the Antarctic cryptoendolithic ecosystem, 
net photosynthesis gain is mostly used to compensate the metabolic cost of survival. 

Scytonemin is a stable and passive sunscreen produced by cyanobacteria that pro- 
tects cells even during long periods of metabolic inactivity (Dillon and Castenholz 
1999). Its synthesis in Chroococcidiopsis sp. has been shown to be modulated by 



128 B. Gomez-Silva et al. 

environmental factors such as UV-A irradiance, temperature, photooxidative 
conditions, and osmotic stress (Dillon et al. 2002). These capabilities, coupled with 
the extreme desiccation resistance of photo synthetic micro-organisms such as 
Chroococcidiopsis, suggest possible models for past life on Mars (Friedmann and 
Ocampo-Friedmann 1995). Given the extreme duration and degree of hyperarid con- 
ditions in the Atacama Desert, the above and other survival and adaptation strategies 
of lithobiont communities from the Atacama should be further evaluated. 



6.5 Patchiness of Transition from High- to Low-Density 
Populations 

As discussed above, the distribution of soil bacteria in the extreme arid core region 
of the Atacama has considerable variability. This is in contrast with the more 
uniform, and higher, density of soil bacteria in the wetter regions of the Atacama. 
The transition between bacteria-rich soils and Mars-like soils is not yet understood. 
We have considered several possibilities: (1) the transition could be gradual with 
the number of organisms and the concentration of organic material dropping off 
monotonically with the decrease in water availability; (2) the transition could be 
sharp, analogous to a tree-line on a mountain slope; (3) the transition could 
be patchy with 'islands' of bacteria-rich soil in otherwise Mars-like terrain. 
Preliminary data suggest that the transition is patchy. 

In the extreme arid core, the soil is dominated by Mars-like conditions with isolated 
islands of bacteria-rich soil. Moving toward wetter regions, these isolated island 
presumably become more numerous and eventually merge to form the continuously 
habitable soils we observe in the regions of the Atacama that receive > 25 mm/yr of 
rain. In the arid core area the patchiness could follow geological patterns in the soils 
or could follow subtle environmental patterns of water availability. Alternatively, the 
patchiness could be an intrinsic response of the microbial ecosystem. Rietkerk et al. 
(2004) have pointed out that self-organized patchiness and the resource concentration 
mechanisms involved have been reported from various ecosystems, but are most 
prominent in arid ecosystems with water as the controlling resource. Typically, this 
observation has been applied to grasses, bushes, and trees in arid regions but may also 
apply to bacteria facing the low water availability of the Atacama. 



6.6 Relevance to Exobiology 

The Atacama Desert, one of the oldest and driest deserts on Earth, provides 
an analogue for life in dry conditions on early or present Mars. We have used 
this analogue to explore the limits of life under Mars-like conditions for hetero- 
trophic bacteria (Navarro-Gonzalez et al. 2003) and for phototrophic cyanobacteria 
(Warren-Rhodes et al. 2006). The core of the Atacama is the end member in 



6 Atacama Desert Soil Microbiology 129 

mineralogical comparisons between Mars and Earth soils and represents soils that 
differ qualitatively from soils in wetter desert environments (Ewing et al. 2006). 

Our biological and chemical results suggest that, if the Viking lander had landed 
in the arid core region of the Atacama, it would have been unable to detect any 
evidence of life. Indeed, the lander instruments would have produced results similar 
to what they produced on Mars. The pyrolysis GCMS would have been unable to 
detect organic material (Navarro-Gonzalez et al. 2006), and the Labeled Release 
experiment would have shown C0 2 release indicating decomposition of added 
organics (Navarro-Gonzalez et al. 2003). Yet there is evidence of life remaining in 
the arid Atacama soils in the form of refractory organic material. These soils can 
be used as an analogue for the development and testing of instruments for organic 
and biological analysis on Mars. In addition, these soils can be used to develop and 
test methods for characterizing the soil oxidant on Mars. Although the presence of 
a soil oxidant has been known in the Atacama now for several years, its nature and 
mechanism of formation is still uncertain. Clearly, we should be able to characterize 
soil oxidants here on Earth before we venture into an examination of Mars 
chemistry. 



6.7 Conclusions: NASA-Supported Research 
in the Atacama Desert 

The relevance of the Atacama Desert to exobiology was appreciated in the 
1960s when R. Cameron from the Jet Propulsion Laboratory collected samples 
from several sites in this hostile environment for use in the Viking testing pro- 
tocols. The microbiological work was done using culturing methods. However, 
at that time very little was known about the heterogeneity of the Atacama due 
to variations in the penetration of the coastal fog as discussed above. The next 
phase of research began in 1994, when NASA and the University of Antofagasta 
established a year-round environmental monitoring station at the University of 
Antofagasta Research site near Yungay. In addition to recording the air temper- 
ature and humidity, rain, dew, light, and wind speed and direction, the unit also 
recorded the soil moisture with two different sensors and at three different 
depths. The results from this station for the years 1994-1998 have been 
published (McKay et al. 2003). The data showed how profoundly dry the 
Atacama was, compared to other deserts that were studied by identical instru- 
ments (e.g., Mojave, Negev, Gobi, etc.). During these same early expeditions 
(ca. 1994, 1996, and 1998), soil samples were collected for microbial analysis 
using culture-dependent and culture-independent techniques from a variety of 
sites around the Atacama. An unexpected lack of bacteria was demonstrated in 
some of the samples. Combined with the results from the environmental moni- 
toring station, indicating that the core region of the Atacama near Yungay was 
50 times drier than the Mojave, these observations led us to focus on this area 
for further investigation. 



130 B. Gomez-Silva etal. 

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GB 190003918 



Chapter 7 

Microbial Communities and Processes in Arctic 

Permafrost Environments 

Dirk Wagner 



7.1 Introduction 

The Arctic plays a key role in Earth's climate system, as global warming is predicted 
to be most pronounced at high latitudes and because one third of the global carbon 
pool is stored in ecosystems of the northern latitudes. Global warming will have 
important implications for the functional diversity of microbial communities in 
these systems. It is likely that temperature increases at high latitudes will stimulate 
microbial activity and carbon decomposition in Arctic environments and accelerate 
climate change by increasing trace gas release (Melillo et al. 2002, Zimov 
et al. 2006). 

In polar regions, huge layers of frozen ground - termed permafrost - cover more than 
25% of the land surface (Zhang et al. 1999) and significant parts of the coastal sea 
shelves (Romanovskii et al. 2005; Fig. 7.1). Permafrost can extend hundreds to 
more than 1,000m into the subsurface (Williams and Smith 1989). This environ- 
ment is controlled by extreme climate and terrain conditions. Particularly, seasonal 
freezing and thawing lead to distinct gradients in temperature and geochemistry in 
the upper active layer of permafrost. As it was thought that these conditions were 
hostile to life, permafrost was considered as uninhabitable even for micro-organisms. 
However, we now know that microbial communities in permafrost environments 
exist and are composed by members of all three domains of life (Archaea, Bacteria, 
and Eukarya), with a total biomass comparable to that of communities of temperate 
soil ecosystems (Wagner et al. 2005). 

The permafrost microbial communities have to overcome the combined action 
of extremely cold temperature, freeze-thaw cycles, desiccation, and starvation 
(Gilichinsky and Wagener 1994; Morozova and Wagner 2007). Recent studies indi- 
cated that micro-organisms not only survive under permafrost conditions, but also 
may sustain active metabolism (Rivkina et al. 2004; Wagner et al. 2007). Although 
methods of modern molecular ecology are still rarely used to study diversity and 



Dirk Wagner 

Alfred Wegener Institute for Polar and Marine Research, Research Unit Potsdam, Telegrafenberg 

A45, 14473 Potsdam, Germany 

e-mail: Dirk.Wagner@awi.de 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 133 

© Springer- Verlag Berlin Heidelberg 2008 



134 



D. Wagner 




] Subsea permafrost extent 
| Continuous permafrost 
| Discontinuous permafrost 
| Sporadic permafrost 
isolated patches 



Fig. 7.1 Terrestrial and submarine permafrost distribution in the northern hemisphere. (International 
Permafrost Association Standing Committee on Data Information and Communication 2003) 



community structure in permafrost environments (for recent examples of such use, 
see Colwell et al. 1999; Rivkina et al. 2000; Wartiainen et al. 2003; Vishnivetskaya 
et al. 2006; Ganzert et al. 2007; Steven et al. 2007), a diverse range of micro-organisms 
have been discovered in the different ecosystems (Shi et al. 1997; Kobabe et al. 
2004; Wagner et al. 2005). Whereas microbial metabolism has been rather well 
studied in temperate environments, little corresponding information is available for 
the Arctic environments. In particular, the importance of microbial diversity for the 
functioning and stability of the Arctic ecosystem, the carbon dynamics controlled 
by micro-organisms, and the reaction of these micro-organisms to changing envi- 
ronmental conditions at high latitudes remain poorly understood. 

In addition to its global relevance as a large carbon reservoir, the permafrost 
extreme environment is of particular interest to astrobiological research as an analogue 
for extraterrestrial permafrost habitats, which are common in our solar system. 
Since the current ESA mission Mars Express detected methane in the Martian 
atmosphere for the first time (Formisano 2004), recent studies have focused on 
methanogenic archaea from permafrost environments as potential candidates for 
life on Mars (Wagner et al. 2001; Morozova et al. 2007; see Chapter 11). 



7 Microbial Communities and Processes in Arctic Permafrost Environments 



135 



This review first describes the environmental conditions in permafrost. It then 
deals with the microbial communities established in these environments, examining 
their function (as far as it is known), and their role and significance in the biogeo- 
chemical cycles. 



7.2 The Permafrost Environment 

Permafrost is defined as ground, comprised of soil, sediment, or rock, and includes 
ice and organic material, that remains at or below 0°C for at least two consecutive 
years (van Everdingen 2005). Arctic permafrost regions are characterized by low 
mean annual air temperatures, low mean annual precipitation (Table 7.1), and poor 
to missing vegetation. During the relatively short period of arctic summer, only the 
surface zone (a few dm thick) of permafrost sediments thaws: this is called the 
active layer. Active layer depths range from a few cm in the high Arctic to more 
than 2 m in subarctic regions. Permafrost can be cemented by ice, which is typical 
for Arctic regions, or, in the case of insufficient interstitial water, may be dry as 
occurs in the Antarctic polar deserts (see Chapter 12) or rocky areas. 

The permafrost environment can be divided into three temperature-depth layers, 
characterized by different living conditions. These are: the active layer, with an 
extreme temperature regime from about +15 to -35°C depending on air tempera- 
ture fluctuations; the upper, perennially frozen permafrost sediments (of 10-20m 
thickness), with smaller seasonal temperature variations of about to -15°C; and 
the deeper permafrost sediments, which are characterized by a stable temperature 



Table 7.1 Climate data for selected localities in circumarctic permafrost environments 

Minimum/ 

Mean Annual Maximum Total 

Temperature Temperature Precipitation 

Locality Coordinates [°C] [°C] [mm] Reference 



Green Harbour, 
Spitzbergen 


78°N, 15°E 


-8 


-19/+6 


370 


French 1996 


Severnay 
Zemlya, 
Krasnoyarsk 


79°N, 91°E 


-14 


-45/+6 


97 


Orvig 1970 


Lena Delta, 
Yakutsk 


73°N, 126°E 


-15 


-48/+ 18 


320 


ROSHYDROMET 
2004 


Lake 

El'gygytgyn, 
Chukotka 


67°N, 172°E 


-10 


-40/+26 


178 


Nolan and 
Brigham- 
Grette 2007 


Dawson City, 
Canada 


64°N, 139°W 


-5 


-31/+14 


343 


French 1996 


Sachs Harbour, 
Canada 


71°N, 125°W 


-14 


-29/+5 


93 


French 1996 



136 



D. Wagner 



regime of about -5 to -10°C (French 1996). The boundary between the active layer 
and the perennially frozen ground is called the permafrost table, which acts as a 
physical and chemical barrier. Intensive physicochemical processes under extreme 
conditions take place in the active layer and upper permafrost sediments (Ostroumov 
2004). In the deeper permafrost layers, conditions have been stable for long periods 
of time and microbial processes are limited (French 1996; Wagner et al. 2007). 

Other components of the specific stratigraphy of permafrost are patterned 
ground formation and various cryogenic structures such as ice wedges, taliks, and 
cryopegs (Fig. 7.2). Thermal conditions determine the presence of such formations 
and structures. For instance, the large differences between summer and winter tem- 
perature in permafrost leads to the formation of typical patterned grounds (e.g., 
sorted circle and high- and low-centered polygons) with a prominent microrelief 
(Fig. 7.3a-c). The development of these structures is often related to the processes 
of ground ice formation. The term "ground ice" describes all types of ice in perma- 
frost deposits, ranging from poor ice crystals to massive horizontal layers of ice 
with a thickness of several decameters. 

Ice wedges occur typically in tundra environments with polygonal patterned 
grounds. In the cold winter season, thermal contraction cracks form polygonal nets. 
These cracks are then filled with snow melt water at the beginning of spring. 
Repeated cracking, filling with water and freezing can produce low -centered 



glacier 




Fig. 7.2 Block diagram of an Arctic permafrost environment showing the different landscape 
units (glacier, tundra, coast, and sea) with the potential cryogenic features (ice complexes and 
wedges, massive ground ice, taliks, cryopegs), differentiated by their thermal regime 



7 Microbial Communities and Processes in Arctic Permafrost Environments 



137 











■ v^^ 












^SKS* 








t.^1 




Mm^ 


. 1 




_ -\ 






_- 




(t) 




Fig. 7.3 Patterned grounds, cryogenic structures, and permafrost soils of Arctic polar regions: (a) 
sorted nets, Dawson City, Canada (photo E.-M. Pfeiffer, University of Hamburg); (b) sorted circle, 
Spitsbergen (photo J. Boike, AWI); (c) low-centered polygons, Lena Delta, Siberia (photo D. 
Wagner, AWI); (d) permafrost soil (Glacic Aquiturbel) of the polygon rim, Lena Delta, Siberia 
(photo L. Kutzbach, University of Greifswald); (e) ice- wedge, Lena Delta, Siberia (photo 
D. Wagner, AWI); (f), ice complex, Lena Delta, Siberia (photo V. Rachold, IASC); (g) permafrost soils 
(Ruptic-Histic Aquiturbel) of an ice complex area, Lena Delta, Siberia (photo D. Wagner, AWI) 



138 D. Wagner 

polygonal microrelief with ice wedges of several meters in width and two to three 
decameters in depth over geological times of tens of thousands of years (Fig. 7.3e; 
Washburn 1978). Pleistocene ice-rich erosional remains of such a polygonal land- 
scape form an ice complex, or Yedoma (Fig. 7.3f). An unfrozen sediment layer or 
body in the perennially frozen ground, located mostly below water bodies, is called 
a talik: this occurs because of local anomalies in thermal, hydrological, hydrogeo- 
logical, or hydrochemical conditions (van Everdingen 2005). Cryopegs (over- 
cooled water brine lenses) are defined as a layer of unfrozen ground that is 
perennially cryotic, forming part of the permafrost (van Everdingen 2005). Freezing 
of cryopegs is prevented by freezing-point depression due to the high salt content 
(140-300 g l- 1 ) of the pore water (Gilichinsky et al. 2005). 

It has been known for some time that the shallow shelves of the Arctic coastal 
seas are underlined by submarine permafrost (see Figs. 7.1 and 7.2), which was 
formed during the Holocene sea level rise by flooding of the formerly terrestrial 
permafrost (Romanov skii et al. 2005). The flooding of the cold terrestrial perma- 
frost (the temperature of which was -5 to -15°C) with relatively warm (-0.5 to 
-2°C) saline sea water changed the system profoundly and resulted in a warming 
of the permafrost (Overduin 2007). 

Permafrost soils (cryosols) have been developed in the upper zone of the cryolithos- 
phere (active layer and upper permafrost sediments) where the temperatures range 
from -50 to +30°C (Yershov 1998). Therefore, permafrost soils are mainly formed 
by cryopedogenesis, which involves freezing and thawing, frost stirring, mounding, 
fissuring, and solifluction. The repeating cycles of freezing and thawing lead to 
cryoturbation features (frost churning) that includes irregular, broken, or involuted 
horizons (Fig. 7.3d) and an enrichment in organic matter and inorganic compounds, 
especially on top of the permafrost table (Van Vliet-Lanoe 1991, Bockheim et al. 
1999). As a result of cryopedogenesis, many permafrost soils are influenced by a 
strong microrelief, which causes small-scale variations in soil types (Fig. 7.3d,g) 
and vegetation characteristics, as well as in the microclimatic conditions. This 
affects the abundance, processes and diversity of microbial communities in per- 
mafrost environments. Table 7.2 summarizes the physiochemical properties of a typi- 
cal permafrost soil, established on the dry rim part of a low-centered polygon from the 
Lena Delta, Siberia. 

The seasonal variation of soil temperature also influences the availability of pore 
water. The presence of unfrozen water is an essential biophysical requirement for 
the survival of micro-organisms in permafrost. As temperatures drop below zero, 
free water is increasingly lost. At the same time, freezing of water leads to an 
increase of salt content in the remaining pore solution. In clayey permafrost soils, 
liquid water was found at temperatures down to -60°C (Ananyan 1970). The most 
important biological feature of this water is its possible role in transfer of ions and 
nutrients (Ostroumov and Siegert 1996). 

Permafrost ecosystems are therefore extremely heterogeneous in nature. They 
are influenced by the regional climatic conditions, which provide harsh and strongly 
fluctuating conditions to their inhabitants. In these habitats, the extraordinarily high 
content of solid components randomly intermixed with gaseous and liquid components 



7 Microbial Communities and Processes in Arctic Permafrost Environments 139 

Table 7.2 Selected physiochemical properties of a permafrost soil (Glacic Aquiturbel) of the 
Lena Delta, northeast Siberia a 

















CH 4 










Depth 






TOC 


TN 


DOC 


jimol 


Sand 


Silt 


Clay 


Horizon b 


[cm] 


T[°C] 


pH 


[%] 


[%] 


[mg H] 


[g" 1 ] 


[%] 


[%] 


[%] 


Ajj 


0-5 


6.4 


n.d. 


2.1 


0.12 


7.3 


0.4 


85.7 


10.4 


3.9 


Bjjgl 


5-12 


5.0 


n.d. 


2.0 


0.11 


7.1 


0.3 


74.3 


20.6 


5.0 


Bjjg2 


12-20 


4.0 


n.d. 


2.4 


0.14 


9.0 


35.3 


68.0 


25.8 


6.3 




20-27 


3.4 


7.9 


3.0 


0.09 


7.3 


65.8 


63.7 


30.3 


6.0 




27-35 


2.4 


6.7 


2.4 


0.07 


4.0 


153.5 


56.5 


34.5 


9.1 


Bjjg3 


35-42 


1.7 


6.8 


2.7 


0.15 


8.7 


224.7 


59.3 


34.0 


6.7 




42-49 


1.0 


n.d. 


3.3 


0.18 


17.3 


478.7 


43.7 


43.8 


12.5 



a Modified from Wagner et al. (2005). 

b Horizon nomenclature according to Soil Survey Staff (1998); T, temperature; TOC, total organic 

carbon; TN, total nitrogen; DOC, dissolved organic carbon. 



hampers the movement of micro-organisms, the mixing of substrates, and physical 
interaction with other organisms. This stimulates the formation of spatially 
separated microcolonies, which are subject to location-based adaptation and micro- 
evolutionary processes. 



7.3 The Permafrost Microbiota 
7.3.1 General Presentation 

The first report on viable micro-organisms in permafrost was given in 1911 by 
Omelyansky. This pioneering investigation was followed by a number of studies 
revealing the presence of significant cell counts and various types of micro-organisms, 
including bacteria, yeasts, fungi, and protozoa, within the active layer and the peren- 
nially frozen ground of permafrost soils (Kris 1940; James and Sutherland 1942; 
Boyd 1958; Boyd and Boyd 1964). Since that time, a number of investigations on 
microbial abundance and physiology within different circumarctic environments have 
been carried out (e.g., Zvyagintsev et al. 1985; Khlebnikova et al. 1990; Rivkina et al. 
2000; Kobabe et al. 2004; Gilichinsky et al. 2005; Zak and Kling 2006; Liebner and 
Wagner 2007). 

Using classical isolation strategies, the most important physiological groups 
of micro-organisms could be recognized, including aerobic and anaerobic heterotrophs, 
methane oxidizers, nitrifying and nitrogen-fixing bacteria, sulfate and iron reducers, ace- 
togens,andmethanogens. The dominantmicrobial genera d£zAcetobacterium,Acinetobacter, 
Arthrobacter, Bacillus, Cellulomonas, Flavobacterium, Methanosarcina, Methylobacter, 
Micrococcus, Nitrobacter, Nitrosomonas, Pseudomonas, Rhodococcus, and Streptomyces 
(e.g., Gilichinsky et al. 1995; Kotsyurbenko et al. 1995; Omelchenko et al. 



140 



D. Wagner 



1996; Shi etal. 1997; Simankova et al. 2000; Suzuki etal. 2001; Wartiainenetal. 2006a). 
Total microbial counts obtained for permafrost soils gave high numbers of micro-organ- 
isms in the range from 10 8 to 10 9 cells g" 1 soil (Kobabe et al. 2004) and for the perennially 
frozen ground between 10 3 and 10 8 cells g" 1 sediment (Rivkina et al. 1998). Table 7.3 
presents some examples of micro-organisms isolated from permafrost environments. 

It is notoriously difficult to obtain a wide diversity of micro-organisms from 
environmental samples in culture, especially from low-temperature habitats, and 
the biogeochemical roles of Bacteria, Archaea, and Fungi have consequently 
been studied using black-box techniques such as epifluorescence direct counts, 
DNA and protein synthesis rates, enzyme activity, and a host of other methods 
that are inherently blind to variations in community composition (e.g., Vorobyova 
et al. 1997; Spirina and Fedorov-Davydov 1998; Bakermans et al. 2003; 
Santruckova et al. 2003; Colwell et al. 1999; Liebner and Wagner 2007; Panikov 
and Sizova 2007). 

Much of what is now known of environmental microbial diversity is based on 
distinguishing between different organisms, as represented by their nucleic acids 



Table 7.3 Examples of micro-organisms isolated from permafrost environments 



Micro-organism Description 



Environment 



Reference 



Aceto bacterium 

tundrae 
Acinetobacter 

strain no. 6 
Candidatus 

Nitrotoga 

arctica 
Carnobacterium 

pleistocenium 
Clostridium 

algoriphilum 

Exiguo bacterium 
strain 255-15 

Methanosarcina 
strain SMA-21 

Methylobacter 
psychrophilus 

Methylobacter 
tundripaludum 

Methylocystis 
rosea 

Psychrobacter 
strain 273-4 



Psychrophilic 

acetogenic bacterium 

Psychrotrophic, 

lipolytic bacterium 

Cold-adapted, nitrite- 
oxidizing bacterium 

Psychrotolerant, 

facultative anaerobe 
Psychrophilic, anaerobic, 

spore-forming 

bacterium 
Psychrophilic, 

osmo-tolerant, 

facultative anaerobe 
Cold-adapted, 

stress-tolerant 

methanogen 
Psychrophilic, 

methane-oxidizing 

bacterium 
Cold-adapted, 

methane-oxidizing 

bacterium 
Cold-adapted, 

methane-oxidizing 

bacterium 
Cold- adapted 

Proteobacterium 



Permafrost soil (tundra 

wetland), Polar Ural 
Permafrost soil (tundra), 

Siberia 
Permafrost soil (polygonal 

tundra), Lena Delta, 

Siberia 
Permafrost (32,000 years 

old), Fox tunnel, Alaska 
Cryopeg (100-120,000 years 

old), Yakutskoe Lake, 

East Siberian sea coast 
Permafrost (2-3 million 

years old), Kolyma- 

Indigirka, Siberia 
Permafrost soil (polygonal 

tundra), Lena Delta, 

Siberia 
Permafrost soil (dwarf birch 

tundra), Northern Russia 

Permafrost soil, Ny-Alesund, 
Svalbard 

Permafrost soil, Ny-Alesund, 
Svalbard 

Permafrost (20-40,000 years 
old), Kolyma-Indigirka, 
Siberia 



Simankova et al. 

2000 
Suzuki et al. 

2001 
Alawi et al. 2007 



Pikuta et al. 2005 

Shcherbakova 
et al. 2005 

Vishnivetskaya 
et al. 2000 

Morozova and 
Wagner 2007 

Omelchenko 
et al. 1996 

Wartiainen et al. 
2006b 

Wartiainen et al. 
2006a 

Vishnivetskaya 
et al. 2000 



7 Microbial Communities and Processes in Arctic Permafrost Environments 141 

or their lipid composition, without actually culturing these organisms or having 
any direct knowledge of their morphology, physiology, or ecology. However, 
modern molecular-ecological studies of diversity and community structure in 
permafrost environments are still rare (for some examples, see Zhou et al. 1997; 
H0j et al. 2005; Neufeld and Mohn 2005; Ganzert et al. 2007; Steven et al. 2007). 

Both with fluorescence in situ hybridization (FISH) and with DNA-based inves- 
tigations, all relevant groups of micro-organisms (alpha, beta, gamma, and delta 
subclasses of the Proteobacteria, Bacteroidetes division, Gram-positive bacteria 
with low and high GC content and Archaea) could be detected at high cell numbers 
in the active layer and in the frozen ground of permafrost (Shi et al. 1997; Zhou 
et al. 1997; Kobabe et al. 2004). Despite differences in the requirements of the 
specific groups, which influence their abundances in the soils, the total diversity 
and quantity of active cells was strongly related to the content and quality of 
organic matter (Kobabe et al. 2004; Wagner et al., 2005). 

In spite of the harsh environmental conditions prevailing in the deeper horizons 
of the active layer close to the permafrost table, there is evidence for a high amount 
of cells (4 x 10 7 cells g" 1 soil) in this zone, that maintain at least minimal activity 
(Kobabe et al. 2004). Detailed bacterial 16S rDNA clone library analyses of a 
polygonal tundra soil from the Lena Delta (northern Siberia) revealed a great 
variability of colonization by representatives of the main phyla (Actinobacteria, 
Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes, Planctomycetes, 
Proteobacteria, and Verrucomicrobia) within the soil of the polygon rim. The com- 
munity composition in the center soil was more homogeneous, although remaining 
influenced by small-scale variations in environmental conditions (S. Liebner, per- 
sonal communication). These communities were dominated by Bacteroidetes, 
Actinobacteria, Proteobacteria, and Firmicutes (in order of decreasing prevalence), 
with a distinct shift along the vertical temperature gradient profile. 

Another study carried out in Northeast Siberia showed that the alpha and delta 
subclasses of the Proteobacteria dominated the microbial community, representing 
a proportion of about 50% of the detected organisms (Zhou et al. 1997). Composition 
of a microbial community from a frozen ground on Ellesmere Island, Canada was 
similar to that of the active layer, but in this case the dominating phyla possessed 
Actinobacteria- and Proteobacteria-related sequences (Steven et al. 2007). 
The archaeal community in this study was composed of 61% Euryarchaeota and 
39% Crenarchaeota, suggesting the presence of a diverse archaeal population. 
In ancient permafrost sediments from Northeast Siberia, the following major 
groups were found: Actinomycetales (Arthrobacter and Microbacteriaceae), other 
Actinobacteria, Bacteroidetes (Flavobacterium), Firmicutes (Exiguobacterium 
and Planomicrobium), Alphaproteobacteria (Sphingomonas), and Gammaproteo- 
bacteria (Psychrobacter and Xanthomonadaceae; Vishnivetskaya et al. 2006). In 
these various studies, a sizable part of the microbial community belonged to thus 
far unclassified micro-organisms, which indicates the existence of large unknown 
communities in permafrost environments. Thus, the physiology and function of 
these presumably dominant micro-organisms remain unknown. 



142 D. Wagner 

7.3.2 Methane-Cycling Micro-Organisms 

Methanogenic archaea and methane-oxidizing bacteria were the object of particular 
attention in permafrost studies, because of their key role in the Arctic methane 
cycle and consequently of their significance for the global methane budget. 

Microbial methane production (or methanogenesis) is a prominent process dur- 
ing the anaerobic decomposition of organic matter. Methanogenesis is solely driven 
by a small group of strictly anaerobic organisms called methanogenic archaea, 
which belong to the kingdom Euryarchaeota (Garcia et al. 2000; see Chapter 9). 

The highest cell counts of methanogenic archaea were detected in the active layer 
of permafrost, with numbers of up to 3 x 10 8 cells g _1 soil (Kobabe et al. 2004). These 
represented between 0.5% and 22.4% of the total cell counts. Phylogenetic analyses 
revealed a great diversity of methanogens in the active layer, with species belonging 
to the families Methanobacteriaceae, Methanomicrobiaceae, Methanosarcinaceae, 
and Methanosaetaceae (H0j et al. 2005; Ganzert et al. 2007; Metje and Frenzel 2007). 
Other sequences detected were affiliated with the euryarchaeotal Rice Cluster II and 
V (Hales et al. 1996; Grosskopf et al. 1998; Ramakrishnan et al. 2001) as well as with 
Group I.3b of the uncultured Crenarchaeota (non-methanogenic archaea; Ochsenreiter 
et al. 2003). 

The detected families were not restricted to specific depths of the soil profiles. 
Environmental sequences from the Laptev Sea coast form four specific permafrost 
clusters (Ganzert et al. 2007). Permafrost Cluster I was recovered mainly from cold 
horizons (with temperatures of less than 4°C) of the active layer and related to 
Methanosarcinacea. Permafrost Clusters II and III related to Methanomicrobiales 
and Permafrost Cluster IV related to Rice Cluster II. It was hypothesized by the 
authors that these clusters comprise methanogenic archaea with a specific physio- 
logical potential to survive under harsh environmental conditions. The phylogenetic 
affiliation of recovered sequences indicated a potential for both hydrogenotrophic 
and acetoclastic methanogenesis in permafrost soils. 

Methanosarcina sp. SMA-21, which is closely related to Methanosarcina mazei, 
was recently isolated from a Siberian permafrost soil in the Lena Delta. The organ- 
ism grows well at 28 °C and slowly at low temperatures (4°C and 10°C) with H 2 / 
C0 2 (80:20, v/v, pressurised at 150kPa) as a substrate. The cells grow as cocci, with 
a diameter of 1-2 |im. Cell aggregates were regularly observed (Fig. 7.4a). 
Methanosarcina SMA-21 is characterized by an extreme tolerance to very low 
temperatures (-78.5°C), high salinity, starvation, desiccation, and oxygen exposure 
(Morozova and Wagner 2007). Furthermore, this archaeon survived for three weeks 
under simulated thermophysical Martian conditions (Morozova et al. 2007; see 
Chapter 12 for a discussion on the possible presence of methanogens on Mars). 

The biological oxidation of methane by methane-oxidizing (or methanotrophic) 
bacteria, which represent very specialized Proteobacteria, is the only sink for methane 
in permafrost habitats (Trotsenko and Khmelenina 2005). Methanotrophic bacteria 
are common in almost all environments, where they can survive under unfavorable 
living conditions through the formation of exospores and cysts. 



7 Microbial Communities and Processes in Arctic Permafrost Environments 



143 




**■ \ 



(b) 












Fig. 7.4 Selected micro-organisms (Bacteria, Archaea) isolated from different permafrost envi- 
ronments: (a) Methanosarcina sp. SMA-21 (D. Wagner and D. Morozova, AWI; bar: 10|im); 
(b) Methylobacter tundripaludum (Wartiainen et al. 2006b; bar: 200 nm); (c) Clostridium algoriphilum 
(Shcherbakova et al. 2005; bar: l|im); (d) Acetobacterium tundrae (Simankova et al. 2000; 
bar: 10|im) (e) Candidatus Nitrotoga arctica (by courtesy of E. Spieck and T. Sanders, University 
Hamburg; bar: 200 nm); (f) Psychrobacter sp. 273-4 (Vishnivetskaya et al. 2000; bar: 5|im) 



144 D. Wagner 

Up to 2 x 10 8 cells of methane-oxidizing bacteria g" 1 soil were detected in 
the active layer of permafrost soils by fluorescence in situ hybridization 
(Liebner and Wagner 2007). Most horizons of the soils were dominated by type-I 
methanotrophic bacteria (see Chapter 9 for a presentation of the various types 
of methanotrophs). Only in samples close to the permafrost table were type-II 
more abundant than type-I methanotrophs. In contrast with this, another study 
using phospholipid fatty acid (PLFA) concentrations and stable isotope probing 
showed that the community growing at low in situ temperatures was dominated 
by type-I methanotrophs (C. Knoblauch pers. communication). This was further 
confirmed by phylogenetic analyses of methanotrophic bacteria in Arctic 
wetland soils of Svalbard, indicating the dominance of type-I over type-II 
methanotrophs. 

Irrespective of whether type-I or type-II methanotrophs are dominant in any 
particular cold location, the analyses revealed the two genera Methylobacter (type 
I) and Methylosinus (type II) in all studied localities (Wartiainen et al. 2003). 
Phospholipid fatty acid analyses revealed the PLFA 18:lAcisl0, a signature for the 
two methanotrophic genera Methylosinus and Methylocystis of the Alphaproteo- 
bacteria, only in the drier sites of polygonal tundra. In contrast, the PLFA 16:lAcis8, 
indicative for the genera Methylomonas, Methylomicrobium, Methylosarcina, and 
Methylosphaera, were detected in all sites of the polygonal tundra in the Lena Delta 
(Wagner et al. 2005). 

Methylobacter psychrophilus, isolated from a Siberian tundra soil, represents a 
cold-loving type-I methane-oxidizing bacterium (Omelchenko et al. 1996). 
Recently, two new species of methanotrophs were isolated from an Arctic wetland 
soil in Svalbard. Methylobacter tundripaludum (Fig. 7.4b) belongs to type I. This 
Gram-negative, rod-shaped, pale-pink pigmented bacterium grows optimally at 
23°C, but with a minimal temperature well down to 10°C (Wartiainen et al. 2006a). 
Methylocystis rosea is a Gram- negative, pink-red pigmented, polymorphic rod 
belonging to type II. It can grow between 5 and 37°C, with optimal growth occurring at 
27°C (Wartiainen et al. 2006b). 



7.3.3 Other Observations on Permafrost Biodiversity 

Some recent studies dealt with the biodiversity of 100,000-120,000 year-old cryopegs 
in Siberian permafrost (Gilichinsky et al. 2005). Direct microbial cell counts revealed 
numbers in the range of 10 7 cells ml -1 saline water. A variety of aerobic and anaerobic, 
sporeless and spore-forming, halophilic and psychrophilic bacteria as well as mycelial 
fungi and yeasts have been isolated, including genera such as Arthrobacter, Bacillus, 
Erwinia, Frigoribacterium, Microbacterium, Psychrobacter, Paenibacillus, Rhodococcus, 
and Subtercola. Clostridium algoriphilum sp. nov. was isolated, and shown to be 
adapted to low nutrient concentrations (Fig. 7.4c; Shcherbakova et al. 2005). The met- 
abolic end products of this anaerobic bacterium are lactate and butyrate, which can be 



7 Microbial Communities and Processes in Arctic Permafrost Environments 145 

used as substrates by heterotrophic Psychrobacter isolates, indicating the possibility of 
a trophic food chain within the microbial communities of cryopegs. 

Additional novel micro-organisms were isolated recently from various habitats. 
For example, Acetobacterium tundrae (DSM 9173) was isolated from tundra wetlands 
of Polar Ural (Simankova et al. 2000). The organism is cold-adapted with a growth 
temperature optimum of 20°C, and a temperature range of 1 to 30°C. It is a Gram- 
positive, oval shaped, flagellated rod (Fig. 7.4d), fermenting H 2 /C0 2 , formate, 
methanol, and several sugars to acetate as the sole end product. Carnobacterium 
pleistocenium, a novel psychrotolerant, facultative anaerobic bacterium, was isolated 
from Pleistocene ice from the Fox tunnel in Alaska (Pikuta et al. 2005). A Gram- 
positive, motile rod, the organism grows best at 24°C, with a range of to 28 °C. 
Metabolic end products are acetate, ethanol, and C0 2 . Exiguobacterium sp. 255-15 
is a nonspore-forming, Gram-positive bacterium isolated from a 2-3 million- 
year permafrost core (Vishnivetskaya et al. 2000). Its cells are short rods about 
1 |im in length with rounded ends. It is a facultative anaerobe but grows most 
profusely aerobically. 

A novel nitrite-oxidizing bacterium was obtained by enrichment culture and 
provisionally classified as "Candidatus Nitrotoga arctica" (Fig. 7.4-e). The organism 
was cultured at 10°C and is characterized by a fatty acid profile which is different 
from those of known nitrite oxidizers but similar to fatty acid profiles of Betaproteo- 
bacteria (Alawi et al. 2007). Psychrobacter sp. 273-4 is a small, non-motile coccoid 
rod (Fig. 7.4f) often found in pairs, isolated from a 20-40 thousand year-old 
Siberian permafrost core (Vishnivetskaya et al. 2000). The strain is characterized by 
rapid growth at low temperatures and excellent survival after exposure to 
long-term freezing. 

Viable green algae were isolated from Arctic deep sediments frozen for 5-7 
thousand years (Vorobyova et al. 1997). All isolates grew slowly at 20-25 °C and 
were sensitive to high light intensities. The photo synthetic pigments chlorophyll a, 
chlorophyll b, and pheophytin were found in a wide range of sediments of different 
genesis and age. 

Both in the active layer and in the perennially frozen sediments, a large variety of 
fungi was detected. In the active layer of Arctic tundra tussock and shrub soils, the 
fungal community was composed of Ascomycota, Basidiomycota, Zygomycota, 
Chytridiomycota, and Glomeromycota (Wallenstein et al. 2007). Although the tus- 
sock communities had higher proportions of Ascomycota (Dothideomycetes, 
Pezizomycetes, and Sordariomycetes), the shrub soils were dominated by Zygomycota 
(Zygomycetes). Another study performed in Alaska reported the dominance of 
basidiomycetous dimorphic yeasts (Mrakia and Leucosporidium) and ascomycetous 
mycelial fungi (Geomyces; Panikov and Sizova 2007). In ancient permafrost deposits 
up to 400,000 years old, only the groups Ascomycota, Basidiomycota, and 
Zygomycota could be detected (Lydolph et al. 2005). 

The absence of a wide spectrum of cultured organisms is a recurrent theme in 
these studies and suggests that many micro-organisms from permafrost environ- 
ments are either unculturable or the appropriate methods of enrichment and cultivation 
have not been used. 



146 D. Wagner 

7.4 Role and Significance of the Microbiota 
7.4.1 Temperature Effects 

Certain key processes of global biogeochemical cycles (e.g., C, N, and S cycles) 
are carried out exclusively by highly specialized micro-organisms (e.g., methano- 
genic archaea, acetogenic, nitrifying, and sulfate-reducing bacteria), which play a 
quantitatively dominant role in mineralization processes (Hedderich and Whitman 
2006; Drake et al. 2006; Bock and Wagner 2006; Rabus et al. 2006). Although the 
physiology and ecology of various micro-organisms from temperate environments 
are well studied, little is known about the activity and function of many of the 
phyla and species in permafrost habitats described in the previous section (see 
Section 7.3). 

The active layer of permafrost is subjected to annual freezing and thawing 
cycles, which result in large temperature and geochemistry gradients along the 
depth profile of the soils. The extreme temperature regime is one of the most impor- 
tant parameters regulating the metabolic activity and survival of micro-organisms. 
Several recent studies demonstrated activities of micro-organisms from the 
active layer and the perennially frozen ground at subzero temperatures. Various 
micro-organisms isolated from Siberian permafrost exhibit metabolic activities 
down to -10°C (Bakermans et al. 2003; Jakosky et al. 2003). The incorpora- 
tion of 14 C -labeled acetate into bacterial lipids examined in microcosm experi- 
ments at temperatures varying between +5°C and -20°C revealed activity of the 
indigenous micro-organisms over this entire range (Rivkina et al. 2000). 

The minimum temperature for growth of micro-organisms was recently 
reported to be -35 °C (Panikov and Sizova 2007). Growth yields of isolated 
micro-organisms similar to those shown above the freezing point were maintained 
down to -17°C. Between -18°C and -35°C, growth was only detectable for three 
weeks after cooling. Then metabolic activity declined to zero, and micro-organisms 
entered a state of reversible dormancy. Studies on methanogenic activity and 
biomass in a Holocene permafrost core from the Lena Delta (Siberia) showed that 
the methane found at certain depths of the sediments originated from modern 
methanogenesis by cold-adapted methanogenic archaea (Wagner et al. 2007). 
These findings are in accordance with the grouping of microbial metabolic rates 
of cold-adapted micro-organisms that was proposed by Price and Sowers (2004): 
rates of the first group are sufficient for microbial growth; those of the second 
group are sufficient for metabolism but too low for growth, whereas rates belong- 
ing to the third group allow survival in a dormant state accompanied by macro- 
molecular damage repair. The reviewed results of microbial metabolism at subzero 
temperatures contradict the idea of the 'community of survivors' in permafrost 
soils (Gounot 1999; Rothschild and Mancinelli 2001), which are not thought to 
'prefer' their environment but are rather said to be more resistant than others that 
were similarly challenged. 



7 Microbial Communities and Processes in Arctic Permafrost Environments 147 

7.4.2 Carbon Cycling 

Currently, the question that is most hotly debated with respect to permafrost eco- 
systems is this one: 'What will happen to the carbon stored in permafrost, in the 
event of a climate change?' The relevance of Arctic carbon reservoirs is highlighted 
by current climate models that predict significant changes in temperature and 
precipitation in the northern hemisphere (Kattenberg et al. 1996; Smith et al. 2002). 
In particular, the degradation of permafrost and the associated release of climate- 
relevant trace gases from intensified microbial turnover of organic carbon and from 
destabilized gas hydrates represent a potential environmental hazard. 

Carbon cycling under anoxic conditions within the predominantly wet permafrost 
soils is mainly performed via methane production, which is the final process in a 
sequence of hydrolysis and fermentation (Schink and Stams 2006). Thus, methano- 
genic archaea stand in close relationship with other micro-organisms of the anaerobic 
food chain, comprising, in particular, acetogenic bacteria, Clostridia, and other bacte- 
ria (Kotsyurbenko et al. 1993; Stams 1994). In cold environments two main pathways 
of energy-metabolism by methanogens dominate: (i) the reduction of C0 2 to CH 4 
using H 2 as a reductant (hydrogenotrophic methanogenesis) and (ii) the fermentation 
of acetate to CH 4 and C0 2 (acetoclastic methanogenesis; Conrad 2005). 

Methanogenic activity was observed at low in situ temperatures with rates of up to 
39nmol CH 4 h _1 g _1 soil in the active layer of permafrost (Wagner et al. 2003; H0j et al. 
2005; Metje and Frenzel 2007). The highest activities were measured in the coldest 
zones of the profiles. Furthermore, it could be shown that the methane production is 
regulated more by the quality of soil organic carbon than by the in situ temperature 
(Wagner et al. 2005; Ganzert et al. 2007). Another important factor affecting archaeal 
communities in permafrost soils is the water regime. Along a natural soil moisture gra- 
dient, changes in archaeal community composition were observed, which suggest that 
the differences in these communities were responsible for the large-scale variations in 
methane emissions observed with changes in soil hydrology (H0j et al. 2006). 

Microbial methane oxidation in the oxic zones of the active layer is of great 
importance to the control of methane releases from permafrost environments. 
Methane-oxidizing bacteria are using methane as their sole carbon source, with 
consequent energy production by the oxidation of CH 4 to C0 2 (Hanson and Hanson 
1996). Methane oxidation rates in Canadian permafrost soils ranged from 58 to 
92% depending on the environmental conditions (Popp et al. 2000). However, the 
methane oxidation activities showed vertical shifts with respect to optimal tempera- 
ture and distribution of type I and type II methanotrophs in Siberian permafrost 
soils (Liebner and Wagner 2007). In the upper active layer, maximum methane 
oxidation potentials were detected at 21°C. Deep active layer zones that are con- 
stantly exposed to temperatures below 2°C show a maximum potential for methane 
oxidation at 4°C. This indicates a dominance of psychrophilic methanotrophs close 
to the permafrost table. 

A close relationship exists between methane fluxes and microbiological processes 
and communities in permafrost soils. Micro-organisms do not only survive in their 



148 D. Wagner 

extreme habitat but also are metabolically active under in situ conditions, which shows 
that the microbial communities are well adapted to low temperatures and extreme 
geochemical gradients. However, they are also tolerant to temperature increases. This 
is evidenced by results showing that a slight temperature increase can lead to a sub- 
stantial increase in methanogenic activity within perennially frozen deposits (Wagner 
et al. 2007). In case of permafrost degradation by thermokarst or coastal erosion proc- 
esses, this would lead to an extensive expansion of the methane deposits and fluxes 
with a subsequent impact on the total atmospheric methane budget. 



7.4.3 Nitrogen Cycling 

Nitrogen turnover is strongly correlated with the carbon cycle but little is known 
about nitrogen fluxes in Arctic ecosystems and the organisms involved. Low tem- 
perature and poor substrate quality often limit decomposition and nitrogen miner- 
alization in many arctic ecosystems (Jonasson et al. 1993). However, higher rates 
of nitrogen fixation were observed in climate change simulation experiments on 
Ellesmere Island, Canada (Deslippe et al. 2005). Nitrifying bacteria were detected 
in permafrost soils and sediments (Bartosch et al. 2002; Alawi et al. 2007). Even in 
old deep permafrost sediments, nitrifiers can survive long periods of starvation and 
dryness (Soina et al. 1991). Nearly nothing is known about the Arctic source 
strength for the long-life greenhouse gases NO and N 2 0. Furthermore, other 
climate-relevant processes such as microbial methane oxidation are influenced by 
the activity of ammonia oxidizers. More generally, Arctic carbon fluxes and turnover 
rates are limited by microbial-mediated nitrogen mineralization. 



7.4.4 Sulfur Cycling 

Sulfur plays a key role in marine biogeochemical cycles, in particular in anaerobic sedi- 
ments of the marine shelfs. About 50% of the carbon mineralization in shelf 
sediments is oxidized via the reduction of sulfate to sulfide by sulfate-reducing 
bacteria (J0rgensen 1982). The released sulfide can be oxidized chemically or by 
sulfide-oxidizing bacteria in aerobic sediment layers. However, coastal erosion and 
sea level rise created the shallow shelves of the Arctic Ocean, for example those of 
the Laptev Sea, the bottom of which corresponds to formerly terrestrial permafrost 
(Rachold et al. 2005; Romanovskii et al. 2005). Flooding of the cold (-5 to -15°C) 
terrestrial permafrost with relatively warm (-0.5 to -2°C) saline, sulfur-rich water 
from the Laptev Sea changed the system profoundly and resulted in a warming of 
the permafrost (Rachold et al. 2007). Studies on the microbial diversity and activity 
in submarine permafrost have been conducted neither by culture-dependent methods 
nor by culture-independent molecular approaches. Therefore, response of microbial 
mineralization and other processes to rising temperatures in these carbon-rich 



7 Microbial Communities and Processes in Arctic Permafrost Environments 149 

permafrost ecosystems, as well as effects on microbial abundance and diversity, are 
totally unknown. 

The permafrost environment forces the adaptation of the microbial communities 
to low temperature conditions and promotes the growth of species that thus far 
remain undetected in temperate ecosystems. Therefore, Arctic permafrost environ- 
ments can be seen as active microbial ecosystems rather than frozen habitats with 
microbial survivors. The evaluation of microbiological data and their correlation 
with climatic and geochemical results represent the basis for the understanding of 
the role of permafrost in the global system. Of particular relevance are feedback 
mechanisms related to nutrient cycles, biogeochemical processes, and greenhouse 
gas emissions in the context of a warming Earth. 



7.5 Conclusions: Future Directions for Research 

Although one fourth of the Earth land surface and distinct areas of the coastal sea 
shelves are affected by permafrost, the physiology, function, and diversity of micro- 
bial communities in these ecosystems is sparsely investigated thus far. This may be 
partially caused by the relative inaccessibility of the investigation areas and the 
associated logistic problems. However, the main difficulty lies in the lack of 
methodologies specific for permafrost sampling and isolation of cold-adapted 
micro-organisms from Arctic soils and sediments. This is shown by the discrepancy 
between the small numbers of psychrophilic micro-organisms isolated thus far from 
permafrost environments in contrast to the observed significant metabolic rates 
under in situ conditions. Methodological developments should consider the following 
aspects: enrichment of micro-organisms should be performed directly in the field 
or in batch or continuous laboratory culture; culture techniques should be developed 
for the enrichment of 'syntrophically associated' micro-organisms; subzero culturing 
methods are needed; and state-of-the-art culture-independent molecular techniques 
for diversity and functional analyses of microbial communities should be applied 
on permafrost. 

The lack of isolates from permafrost also limits possible biotechnological uses. 
Cold-adapted micro-organisms from permafrost exhibit properties very different 
from those of other thermal classes. Therefore, the vast genetic resources of 
micro-organisms from permafrost environments remain nearly unexploited. It is 
likely that mainly extremophilic microbes could offer technologically and/or eco- 
nomically significant products such as enzymes, polysaccharides, osmoprotectors, 
and liposomes (Cavicchioli et al. 2002). Therefore, one essential goal of microbial 
diversity exploration in cold regions will be to recover new isolates, some of which 
will prove useful for biotechnology processes or medicine. 

Apart from the global relevance of permafrost as a large carbon reservoir, this 
extreme environment is also of particular interest to astrobiological research, as an 
analogue for extraterrestrial permafrost habitats, which are a common occurrence in 
our solar system (Gilichinsky 2001; Wagner et al. 2001). Particularly, the observation 



150 D. Wagner 

of methane in the Martian atmosphere by the current mission of the European Space 
Agency, Mars Express (Formisano 2004), has stimulated the debate over possible 
microbial life on Mars (see also Chapter 11). Recently, it has been shown that metha- 
nogenic archaea isolated from Siberian permafrost environments are more tolerant to 
environmental stress and simulated thermophysical Martian conditions than metha- 
nogens from temperate ecosystems (Morozova and Wagner 2007, Morozova et al. 
2007). Micro-organisms from terrestrial permafrost are valuable model organisms in 
our effort to investigate the possibility of microbial life in extraterrestrial permafrost 
ecosystems. 



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Chapter 8 

Aerobic, Endospore-Forming Bacteria 

from Antarctic Geothermal Soils 

Niall A. Logan(K) and Raymond N. Allan 



8.1 Introduction: Taxonomy of Bacillus Species 
and Related Genera 

The term 'aerobic endo spore-forming bacteria' is used to embrace Bacillus species 
and related genera, for which the production of resistant endospores in the presence 
of oxygen remains the defining feature. They are also expected to possess Gram- 
positive cell wall structures (but staining reactions, even in young cultures, may be 
Gram- variable or frankly Gram-negative), and may be aerobic or facultatively 
anaerobic. These characters have formed part of the definition of the group for 
many years, but some exceptions have emerged. Bacillus infernus and B. arseni- 
ciselenatis are strictly anaerobic, and spores have not been detected in B. infernus, 
B. subterraneus, and B. thermoamylovorans . 

Molecular taxonomic methods have had a huge impact on the classification of 
these organisms, and the number of taxa, including thermophiles, has increased 
greatly. The 1986 edition of Bergey's Manual of Systematic Bacteriology (Claus 
and Berkeley 1986) listed 40 valid Bacillus species, of which only four were true 
thermophiles. These were Bacillus stearothermophilus, B. acidocaldarius, and 
B. schlegelii, each with strains reported from geothermal soils (see Table 8.1), and 
B. thermoglucosidasius. "Bacillus caldolyticus " , "B. caldotenax", and "B. cal- 
dovelox" (Heinen and Heinen 1972) were isolated from naturally heated waters and 
were listed as 'Species incertae sedis.' These still await validation. 

Since that 1986 edition of Bergey's Manual and up to late 2006, 229 further species 
have been newly described or revived among Bacillus and the 1 1 genera derived 
from it (subsequently two of these new genera were merged, so there are now 10 new 
genera recognized). Furthermore, 20 new genera containing 59 species have been 
proposed to accommodate novel aerobic endospore formers not previously assigned 
to Bacillus. Overall, then, there have been proposals for 30 new genera and 288 new 



Niall A. Logan 

Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens 

Road, Glasgow G4 0BA, United Kingdom 

e-mail: nalo@gcal.ac.uk 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 155 

© Springer- Verlag Berlin Heidelberg 2008 



156 



N.A. Logan and R.N. Allan 



Table 8.1 Thermophilic, aerobic endospore-forming bacteria from geothermal sources 
Genus and Species of 
Organism (and Number 
of Species in Genus) 



Original Reference 



Original Source 3 



Other Sources 3 b 



Geobacillus (17) 

G stearothermophilus 



Donk 1920 



Canned corn and beans 



G gargensis 
G. tepidamans 



Nazina et al. 2004 
Schaffer et al. 2004 



G thermodenitrificans Ambroz 1913 



Hot spring 

Geothermal soil, 
Yellowstone, USA, 
beet sugar factory 

Soil 



G thermoleovorans Zarilla & Perry 1987 Soil, muds, sludge 



Food, milk, water, 
soil, hot com- 
post, sugar beet 
juice, hot spring, 
geothermal soil, 
Southern Urals 
(Golovacheva et al. 
1965), hydrother- 
mal vents 



Uncultivated soil, 
sugar beet juice, 
hot compost, 
geothermal soil, 
South Sandwich 
Islands (Logan 
et al. 2000), hydro- 
thermal vents 

Uncultivated soil, 
hydrothermal 
vents, petroleum 
reservoirs, hot 
springs 



"G thermoleovorans 
subsp. strombol- 
iensis " 

G. vulcani 

Geobacillus spp. 

Bacillus (134) 
B. aeolius 
B. coagulans 



Romano et al. 2005 



Geothermal soil, Eolian 
Islands, Italy 



Caccamo et al. 2000 Hydrothermal vent 



Gugliandolo et al. 2003 Shallow marine vent 
Hammer 1915 Evaporated milk 



B. fumarioli 



B. infernus 



Logan et al. 2000 Geothermal soil, 

Antarctica 

Boone et al. 1995 Deep terrestrial 

subsurface 



Deep-sea hydrothermal 
vents, sea mud 



Soil, canned foods, 
tomato juice, 
gelatin, milk, 
medical prepara- 
tions, silage, 
geothermal soil, 
Southern Urals 
(Golovacheva et al. 
1965) 

Gelatin 



(continued) 



8 Aerobic, Endospore-Forming Bacteria from Antarctic Geothermal Soils 



157 



Table 8.1 (continued) 



Genus and Species of 

Organism (and Number 

of Species in Genus) Original Reference 



Original Source 3 



Other Sources 3 



B. schlegelii 



B. thermantarcticus 

B. tusciae 
Alicyclobacillus (11) 

A. acidocaldarius 



A. acidocaldarius 

subsp. rittmannii 
A. hesperidum 
A. vulcanalis 
Alicyclobacillus sp. 



Aneurinibacillus (5) 

An. terranovensis 

Anoxybacillus (10) 

Anox. amylolyticus 

Anox. ayderensis 
Anox. gonensis 
Anox. flavithermus 
Anox. kestanbolensis 
Anox. voinovskiensis 
Brevibacillus (13) 
Br. levickii 

Caldalkalibacillus (1) 

C. thermarum 
Sulfobacillus (4) 

Sulfobacillus spp. 



Vulcanibacillus (1) 

Vulc. modesticaldus 



Schenk & Aragno 1979 Lake sediment 



Nicolaus et al. 1996 Geothermal soil, 

Antarctica 

Bonjour & Aragno 1984 Geothermal pond 



Darland& Brock 1971 



Hot acid springs, acid 
geothermal soil, 
Hawaii 



Geothermal water, mud 
and ash, glacier 
ice, air, Antarctic 
geothermal soil 

(Hudson et al. 1988) 



Antarctic geother- 
mal soil (Hudson 
& Daniel 1988), 
gelatin 



Nicolaus et al. 1998 Geothermal soil, 

Antarctica 
Albuquerque et al. 2000 Geothermal soil, Azores 

Simbahan et al. 2004 Hot spring 



Antarctic geothermal 
soil (Bargagli et al. 
2004) 



Allan et al. 2005 



Poli et al. 2006 

Dulger at al. 2004 
Belduz et al. 2003 
Heinen et al. 1982 
Dulger at al. 2004 
Yumoto et al. 2004 

Allan et al. 2005 



Xue et al. 2006 



Geothermal soil, 
Antarctica 

Geothermal soil, 
Antarctica 

Hot spring 
Hot springs 
Hot spring 
Hot spring 
Hot spring 

Geothermal soil, 
Antarctica 

Hot spring 



Geothermal waters, 
Yellowstone 
National Park, 
USA, Montserrat 
Island 



L'Haridon et al. 2006 



Deep-sea hydrothermal 
vent 



a Sources of organisms corresponding to geothermal soils are indicated in boldtype. 
b References for isolation from geothermal soil are given. 



158 N.A. Logan and R.N. Allan 

or revived species or new combinations, and yet only seven proposals for merging 
species or subspecies were made in that time. Nearly 50 of these new species are 
thermophiles, and several moderate thermophiles have also been described, but 
rather few have been isolated from geothermal soils (see Table 8.1). 



8.2 Habitats of Thermophiles 

Most aerobic endospore formers are saprophytes widely distributed in the natural 
environment, but some are opportunistic or obligate pathogens of animals, including 
humans, other mammals, and insects. The main habitats are soils of all kinds, ranging 
from acid to alkaline, hot to cold, and fertile to desert, and the water columns and 
bottom deposits of fresh and marine waters. The bacteriology of geothermal soils 
has received much less attention than that of hot springs and pools, hydrothermal 
vents, and other heated aqueous environments. There is a long history of studies on 
thermophilic Bacillus, dating from the proposal of the best-known species, B. 
stearothermophilus (now Geobacillus stearothermophilus), from canned food in 
1920. Of the present 17 valid species in Geobacillus, there are reports of eight with 
isolates from unheated soils, six from aqueous environments associated with oil or 
gas fields, six from composts, five from hot springs and shallow hydrothermal 
vents, four from foods, including canned foods, milk, and sugar processing, and 
only three (G. stearothermophilus, G. tepidamans, and G. thermodenitrificans) 
from geothermal soils (see Table 8.1). 

A further taxon from geothermal soil, "G thermoleovorans subsp. stromboliensis" , 
awaits validation. Further, unidentified, thermophilic endospore formers have been 
reported from hydrothermal vents, geothermal soils, and sea muds (White 
et al. 1993; Marteinsson et al. 1996; Takami et al. 1997). Although the strains of 
White et al. (1993) were from several geothermal sites around the world, including 
Iceland, it is not clear which of their isolates were from heated soils. Other ther- 
mophiles are found in the genera Alicyclobacillus, Aneurinibacillus, Anoxy bacillus, 
Bacillus, Brevibacillus, Caldalkalibacillus, Sulfobacillus, Thermobacillus, Ureibacillus, 
and Vulcanibacillus, but of the newer thermophilic taxa of aerobic endospore 
formers, fewer than 20% include strains that have been isolated from geothermal 
soils, and it is remarkable that over half of those were first isolated from Antarctic 
geothermal soils (see Table 8.1). 

Although thermophilic aerobic endospore formers and other thermophiles might 
be expected to be restricted to hot environments, they are also widespread in cold 
environments and appear to be ubiquitously distributed in soils worldwide. Indeed, 
Weigel (1986) described how easy it is to isolate such organisms from cold soils 
and even from Arctic ice. Endospores readily survive distribution from natural 
environments to a wide variety of other habitats, as the example of B. fumarioli, 
described in detail below, demonstrates. 

Strains of Geobacillus with growth temperature ranges of 40 to 80°C can be 
isolated from subsurface layers of soils whose temperatures never exceed 25 °C 



8 Aerobic, Endospore-Forming Bacteria from Antarctic Geothermal Soils 



159 



(Marchant et al. 2002). That spores may survive in such cool environments without 
any metabolic activity is understandable, but their wide distribution and contribu- 
tion of up to 10% of the cultivable flora suggest that they do not merely represent 
contamination from hot environments (Marchant et al. 2002). A study of some Irish 
temperate soils found aerobic thermophile counts of 1.5-8.8 x 10 4 colony-forming 
units per gram, and similar results were obtained for other temperate soils from 
Europe (McMullan et al. 2004), suggesting that these are part of the autochthonous 
flora (Rahman et al. 2004). It is possible that the direct heating action of the sun on 
the upper layers of the soil, and local heating from the fermentative and putrefactive 
activities of mesophiles, might be sufficient to allow the multiplication of ther- 
mophiles, but perhaps these organisms are capable of very low levels of activity at 
normal environmental temperatures (McMullan et al. 2004). 



8.3 Antarctic Geothermal Soils 

There was constant volcanic activity in Antarctica during the Cenozoic period, and 
steaming ground is to be found in a number of circumpolar islands and on the con- 
tinent (Fig. 8.1). Thus, although Antarctica is largely an ice-bound continent that 



Bouvetoya 



CANDLEMAS ISLAND 

.<r .* South Sandwich 

.- Islands 

Antarctic Circle 



Marion Island 



- WW 




lies Kenguelen ft 
Heard Island 

WE - 



1000 km 



Fig. 8.1 Map of Antarctica and the sub- Antarctic islands, with geothermal sites named. Names 
in capital letters indicate the sites from which Bacillus fumarioli, Brevibacillus levickii and 
Aneurinibacillus terranovensis were isolated 



160 N.A. Logan and R.N. Allan 

relies upon solar heating during the summer to support a sparse growth of terrestrial 
life, several sites exist where volcanic activity warms the soil and steam emissions 
from fumaroles condense to maintain relatively steady water supplies that may sup- 
port the growth of vegetation. All of these places are remote, and are costly and 
difficult to visit, and therefore no comprehensive study of the microbiologies of 
their geothermal soils has been made, but we are fortunate to have some informa- 
tion on the aerobic endosporeforming floras of five of these sites. 



8.3.1 The Antarctic Continent 

Three sites, Mt Erebus, Mt Melbourne, and Mt Rittmann, are the only known high- 
altitude localities of fumarolic activity and associated vegetation within Antarctica. 
The unique selective pressures of such sites make the organisms that live there of 
special biological interest (Broady 1993), and they can harbour unique vegetation 
communities which appear to have formed following colonisation by propagules 
from circumpolar continents (Linskens et al. 1993). 

Mounts Erebus and Melbourne represent two of the four provinces of the 
McMurdo Volcanic Group, which is one of the most extensive alkali volcanic prov- 
inces in the world. These two volcanoes were named in 1841 by James Clark Ross 
after his expedition ship Erebus, and the British Prime Minister Lord Melbourne. It 
is worth noting in passing that some of the expedition's specimens were submitted 
to the eminent botanist Christian Ehrenberg, in Berlin, for microbiological 
examination. It was Ehrenberg who had proposed Vibrio subtilis (now Bacillus 
subtilis, and the type species of the genus) in 1835. 

Mt Erebus (3,794m; 77° 32'S; 167° 8'E) is the most active volcano on the 
Antarctic continent, and the largest of four cones on Ross Island. Its cone rises 200 
to 300 m above the summit plateau and it is bordered by a side crater, which is the 
rim of a filled-in older crater or caldera, to the SW. The floor of the main crater has 
an inner crater, whose floor bears a fumarolic ridge. The walls of the main crater 
also bear fumaroles, and discontinuous lines of fumaroles and ice towers radiate 
from the crater rim and across the plateau. Patches of warm ground lie on the rim 
of the main crater, within the side crater, and on the plateau. An area of warm 
ground to the NW of the main crater, with surface ground temperatures that may 
reach 75 °C, is an Antarctic Specially Protected Area (ASPA) called Tramway 
Ridge; the ice-free ground here is terraced, and the steep sides of the terraces bear 
the main crusts of vegetation. 

Mt Melbourne (2,733 m; 74° 21'S; 164° 42'E) is situated in the centre of a rela- 
tively young (probably 2-3 x 10 6 years old) volcanic field that has been formed by 
a large number of small, individual eruptive centres (Broady et al. 1987). Located 
on the southern rim of the main summit crater of Mt Melbourne is an ASPA called 
Cryptogam Ridge (Fig. 8.2). This is a deglaciated site with soil temperatures typi- 
cally reaching 40-50°C at depths of a few centimetres; it supports a unique com- 
munity including algal and bryophyte species unknown elsewhere in Antarctica 



8 Aerobic, Endospore-Forming Bacteria from Antarctic Geothermal Soils 



161 




Fig. 8.2 Mount Melbourne, Northern Victoria Land, Antarctica, looking towards Wood Bay in 
the Ross Sea. The deglaciated Antarctic Specially Protected Area called Cryptogam Ridge is seen 
in the foreground. The icy hummock in the centre of the picture is formed by condensate freezing 
above a fumarole 




Fig. 8.3 Mount Rittmann, Northern Victoria Land, Antarctica, showing areas of heated soil with 
fumaroles, and ice towers formed from frozen fumarole condensate, interspersed with patches of 
permafrost 



(Nicolaus et al. 1991). The flora of the geothermally heated area of the northwest 
(NW) slope of the mountain, lying at 2,400 to 2,500 m, is less well developed than 
that of Cryptogam Ridge. 

Mt Rittmann (2,600m; 73° 27'S; 165° 30'E) (Fig. 8.3) was discovered during the 
fourth Italian Antarctic Expedition (1988-1989). The soil surface temperature 



162 N.A. Logan and R.N. Allan 

ranges from 34.4-41.5°C and there is patchy development of vegetation. The 
geothermally heated biosystem at Mt Rittmann has been described by Bargagli 
et al. (1996). It lies at an altitude of about 2,600m, and has small fumaroles whose 
internal temperatures (at 10 cm depth) range between 50°C and 63 °C; patches of 
moss grow on the warm soil which has a relatively high moisture content (by Antarctic 
standards) and a pH of around 5.4. 

Soil samples were collected from the NW slope of Mt Melbourne and from the 
Mt Rittmann geothermal site by British Antarctic Survey (BAS) members of the 
international BIOTEX 1 expedition during the 1995-1996 austral summer, and 
further samples were taken from these sites and from Cryptogam Ridge during the 
1998-1999 austral summer by one of the authors (NAL). 

Although Mt Melbourne and Mt Rittmann lie in the same volcanic province, and 
their soils may appear similar in some respects (they have very low concentrations 
of essential nutrients such as N and P), Bargagli et al. (1996) showed that there 
were differences in the mineral contents of soil samples collected from Mt 
Melbourne (higher Cu and Zn) and Mt Rittmann (higher Cd and Pb), and Pepi 
et al. (2005) reported that mossy and unvegetated soil samples from the NW slope 
of Mt Melbourne had higher iron contents than did soils from Cryptogam 
Ridge and Mt Rittmann. For other major elements, soils from the NW slope 
of Mt Melbourne also had the highest contents of Na and Al; soils from Cryptogam 
Ridge had the highest content of Mg; and soils from Mt Rittmann had the lowest 
contents of Ca, Al, and Fe. For trace elements, soils from the NW slope of 
Mt Melbourne also had the highest contents of Cd, Cr, Pb, and Zn; soils 
from Cryptogam Ridge had the highest contents of Cu and Hg; and soils from 
Mt Rittmann had the lowest contents of Cu and Hg. Thus organisms in these soils 
may be constrained by low concentrations of essential nutrients and, in some cases, 
relatively high concentrations of toxic minerals. 



8.3.2 The South Sandwich Archipelago 

The South Sandwich archipelago comprises 1 1 islands, all of which exhibit recent 
or continuing volcanic activity. They lie on the Scotia Ridge between latitude 
56°18' and 59°28 / S and longitude 26°14 / and 28°11'W; this volcanic arc has a 
deep-sea trench on its convex (eastern) side descending to over 7,000m. The fauna 
and flora of Candlemas Island (Fig. 8.4; map in Logan et al. 2000) are more exten- 
sive than those of other islands in the arc, and include penguins, petrels, skuas, and 
seals, grass, bryophytes, and lichens. The southern massif forms the largest part of 
the island, and is an ice-capped remnant of an extinct volcano. In the younger and 
actively volcanic northern part lies Lucifer Hill (232m; 57°04'S, 26°42'W), a 
complex of scoria cones surrounded by a mass of five main lava flows. The oldest 
of these bear ash mantles, whereas the youngest is very recent. There are patches 
of moss around active fumaroles high on the hill, around inactive fumaroles at 
Clinker Gulch, and on lava soils at the base of the volcanic cone, with temperatures 



8 Aerobic, Endospore-Forming Bacteria from Antarctic Geothermal Soils 



163 




Fig. 8.4 Candlemas Island, South Sandwich Archipelago, viewed from the east. Lucifer Hill, in 
the unglaciated northern part of the island, is seen on the right-hand side of the picture, with its 
summit partially obscured by cloud. (Aerial photograph kindly provided by Dr John L. Smellie, 
British Antarctic Survey. © British Crown Copyright/MOD.) 



ranging from 85°C down to 0°C. The northern and southern parts of the island are 
linked by an area of low flat sand containing two large lagoons. Mossy soil samples 
were collected from the summit and base of Lucifer Hill by British Antarctic 
Survey personnel on behalf of one of the authors (NAL) during the 1996-1997 
austral summer. 



8.3.3 Deception Island 



Deception Island (62°57'S, 60°38'W) is one of seven islands that comprise the 
South Shetland archipelago. It is 17-km across and ring-shaped, being the rim of a 
caldera that has been flooded by the sea, and it lies on the expansion axis of the 
Bransfield Rift that separates the archipelago from the Antarctic Peninsula. A nar- 
row gap in the caldera rim leads to the basin of Port Foster, a perfect natural 
harbour. It has seen human activity since the 1820s, including sealing, whaling, and 
scientific research; however, there were major eruptions in 1967, 1969, and 1970, 
and the island is less frequented now. Several ASPAs have been established, and the 
whole island is an Antarctic Specially Managed Area. It was visited by a Spanish 
scientific expedition in the austral summer of 1989-1990, and water and sand sam- 
ples were collected from four geothermal sites: a fumarole at Cerro Caliente, Irizar 
Lake, Kroner Lake, and Whalers Bay (Llarch et al. 1997). 



164 N.A. Logan and R.N. Allan 

8.4 Endospore Formers from Antarctic Geothermal Soils 

The first report of an aerobic endospore former isolated from Antarctica was by 
Dr A. L. McLean, who studied the bacteriology of ice and snow during Douglas 
Mawson's Australasian Antarctic Expedition of 1911-1914. Little other Antarctic 
bacteriology was published until Darling and Siple (1941) described the isolation 
of 178 strains from various Antarctic environments, and found 66% of them to 
belong to nine Bacillus species. Ugolini and Starkey (1966) isolated bacteria from 
fumaroles of Mt Erebus, but according to Hudson and Daniel (1988) these were 
probably mesophiles. 

The first isolations of thermophilic endospore formers were made by Hudson 
and Daniel (1988) from Tramway Ridge and its locality, and from the side and west 
craters of Mt Erebus. They found strains of Bacillus in most of their samples. In particu- 
lar, they isolated strains resembling B. acidocaldarius (now in Alicyclobacillus) in 
the more acid soils of Tramway Ridge and its neighborhood (Hudson et al. 1989), 
Bacillus schlegelii (capable of utilizing thiosulfate; Hudson et al. 1988) in an acid 
soil near Tramway Ridge, and anaerobes resembling Clostridium thermohydrosul- 
furicum (now Thermo anaerob act er thermohydrosulfuricus) from most of their 
samples. Bacillus schlegelii was first isolated from the sediment of a Swiss lake, 
and B. tusciae was discovered when Bacillus schlegelii was first sought in geother- 
mal environments (Bonjour and Aragno, 1984). 

Nicolaus et al. (1991) isolated four strains of thermophilic eubacteria from 
Cryptogam Ridge, Mt Melbourne, and one strain from near the seashore at the foot 
of the mountain, and the effects of growth temperature on polar lipid patterns and 
fatty acid compositions of these and other strains were compared (Nicolaus et al. 
1995). One of the Cryptogam Ridge isolates was proposed as Bacillus thermoant- 
arcticus (Nicolaus et al. 1996). Its exopoly saccharide chemistry has been investi- 
gated (Manca et al. 1996) and its digestion of xylan studied (Lama et al. 2004). The 
proposal was later validated and the name of the isolate corrected to Bacillus 
thermantarcticus. However, it should probably belong in Geobacillus. 

Nicolaus et al. (1998) also isolated strains of Alicyclobacillus from Mt Rittmann, 
and proposed the new subspecies, Alicyclobacillus acidocaldarius subsp. rittmannii; 
strains had co-cyclohexyl fatty acids, MK-7 quinones, and hopanoids characteristic of 
Alicyclobacillus species, but lacked amylolytic activity. The effects of growth tem- 
perature on lipid modulation were studied in detail (Nicolaus et al. 2002). In 30 geo- 
thermal soil samples from Mt Rittmann, these authors found only aerobic endospore 
formers belonging to Alicyclobacillus; it will be recalled (see above, this section) that 
Hudson et al. (1989) found strains similar to Alicyclobacillus (then Bacillus acido- 
caldarius) on Mt Erebus. Bargagli et al. (2004) isolated a strain tentatively identified 
as an Alicyclobacillus species from the iron-rich NW slope of Mt Melbourne, and 
found that it needed iron supplements in its growth media (Pepi et al. 2005). 

Llarch et al. (1997) studied six isolates from sand, sediment, and water from geo- 
thermal sites on Deception Island. None of these strains was identifiable as a member 
of an established thermophilic species of Bacillus, but two strains from fumarolic 



8 Aerobic, Endospore-Forming Bacteria from Antarctic Geothermal Soils 



165 



water showed some relationship with B. (now Geobacillus) stearothermophilus . 
More interesting was the identification of two strains as B. licheniformis and B. 
megaterium, and of two others as outliers of the species B. firmus and B. lentus; all 
four species are known as mesophiles, but the Deception Island isolates all had 
optimal growth temperatures between 60 and 65 °C, which considerably extend the 
maximum growth temperatures known for these species. 

Logan et al. (2000) examined geothermal soil samples from Mt Melbourne (both 
Cryptogam Ridge and the NW slope), Mt Rittmann, and Candlemas Island. They 
used a variety of growth conditions in order to isolate aerobic endospore formers 
but they did not find strains of Bacillus thermantarcticus or Alley clobaciilus acido- 
caldarius subsp. rittmannii. Instead, they isolated an organism that grew optimally 
at pH 5.5 and 50°C on a nutritionally weak, solid medium (Bacillus fumarioli agar, 
or BFA: 0.4% yeast extract, 0.3% KH 2 P0 4 , 0.2% (NH 4 ) 2 S0 4 , and traces of MgS0 4 
and CaCl 2 ; with 5mg/l MnS0 4 to enhance sporulation). The organism subsequently 
grew and sporulated better on a medium of half this nutrient strength. The organism 
was isolated from Cryptogam Ridge on Mt Melbourne (but not the NW slope of Mt 
Melbourne), and from Mt Rittmann and Candlemas Island and was proposed as 
Bacillus fumarioli (Fig. 8.5). It was found on Mt Rittmann both as spores and veg- 
etative cells, in soils whose temperatures ranged from 3.4°C to 62.5°C, the propor- 
tions of sporulated cells tending to be higher at the temperature extremes (9% at 
8.3°C; 29% at 58.5°C), and lower (3% at 42.5°C) at temperatures approaching the 
growth optimum. 

Finding B. fumarioli on two volcanoes 110 km apart and on Candlemas Island, 
which is about 5,500km distant from Mt Melbourne, was quite striking, and it 
seemed remarkable that it could not be isolated from the NW slope of Mt Melbourne, 




Fig. 8.5 Photomicrograph of sporangia and vegetative cells of Bacillus fumarioli viewed by 
phase contrast microscopy; ellipsoidal and cylindrical spores lie paracentrally and subterminally 
in unswollen sporangia. Bar represents 2 urn 



166 N.A. Logan and R.N. Allan 

despite repeated sampling; nor was it isolated from 25 cold soils local to 
Mt Melbourne. It was even more surprising, therefore, that using BFA De Clerck et al. 
(2004) were able to isolate B. fumarioli from gelatin production plants in Belgium, 
France, and the USA. Although both ecosystems have similarly low pH and moderately 
high temperatures, their geographical separations are huge, and the organic load in 
Antarctic soils is very low compared with that of gelatin. A polyphasic taxonomic 
comparison showed very close relationships between the Antarctic and gelatin 
isolates; the latter organisms did, however, produce an abundant protein with high 
similarity to a stress response protein, and subtractive DNA hybridization revealed 
genomic differences between the two sets of isolates that might indicate adaptive 
evolution to a specific environment (De Clerck et al. 2004). 

It is of special interest that the NW ridge of Mt Melbourne failed to yield 
B. fumarioli from samples taken on two occasions. Why this particular geothermal 
site, lying a short distance from Cryptogam Ridge, should not yield the organism is 
not understood. It was noted that moss was absent from the NW ridge, yet 
B. fumarioli was isolated from both mossy and moss-free areas of Cryptogam Ridge 
and Mt Rittmann. Broady et al. (1987) remarked on the low diversity of Victoria 
Land warm ground bryophytes compared with Deception Island and the South 
Sandwich Islands in the maritime Antarctic, and suggested that as none of the local 
cold-ground bryophytes of Victoria Land had colonized the local volcanoes, it may 
be inferred that the soil chemistries of the fumarole environments might be unsuit- 
able; indeed, as noted above, Bargagli et al. (1996) and Pepi et al. (2005) found 
appreciable differences in the mineral contents of these different soils. Broady et al. 
(1987) also noted that the geothermal areas of Mt Erebus and Mt Melbourne are, in 
comparison with maritime Antarctica, much farther from the rich propagule sources 
of more temperate lands to the north and west, and well south of the circumpolar 
westerly airstream that might carry and deposit such propagules; however, the dis- 
covery of B. fumarioli in Europe and America suggests that this organism is widely 
dispersed. Llarch et al. (1997) did not find B. fumarioli in their geothermal soils 
from Deception Island, but they did not cultivate at pH 5.5 from their samples. 

Two other kinds of aerobic endospore formers were isolated from Mt. Melbourne 
during the expedition of 1998-1999 (Logan et al. 2000). They were scanty and dif- 
ficult to cultivate, and so were not studied further at that time, but additional strains 
were isolated from the same soil samples back in Glasgow. Allan et al. (2005) sub- 
jected 13 such strains to polyphasic taxonomic study, and proposed seven isolates 
from the NW slope of Mt Melbourne as the new species Brevibacillus levickii (Fig. 
8.6), and proposed six isolates from Cryptogam Ridge and the vents and summit of 
Mt Rittmann as another new species, Aneurinibacillus terranovensis (Fig. 8.7). 
Brevibacillus strains were not isolated from the sites at Mt Rittmann or Cryptogam 
Ridge and Aneurinibacillus strains were not isolated from the NW slope of Mt 
Melbourne. The distribution of An. terranovensis thus correlates with that of B. 
fumarioli on Mts Melbourne and Rittmann, whereas Br. levickii was only found in 
soils of the NW slope of Mt Melbourne, from which An. terranovensis and B. 
fumarioli could not be isolated; perhaps these observations owe something to the 
differences in soil chemistries that Bargagli et al. (1996) and Pepi et al. (2005) 
discovered at these sites. 



8 Aerobic, Endospore-Forming Bacteria from Antarctic Geothermal Soils 



167 




Fig. 8.6 Photomicrograph of sporangia and vegetative cells of Brevibacillus levickii viewed by 
phase-contrast microscopy; ellipsoidal spores lie subterminally and terminally in swollen spor- 
angia. Bar represents 2|im 




Fig. 8.7 Photomicrograph of sporangia and vegetative cells of Aneurinibacillus terranovensis 
viewed by phase-contrast microscopy; ellipsoidal spores lie centrally, paracentrally and subtermi- 
nally in swollen sporangia. Bar represents 2jim 



168 N.A. Logan and R.N. Allan 

Our emphasis thus far has been on thermophilic or thermotolerant organisms, 
but the same geothermal soils have also yielded mesophilic species. Bargagli et al. 
(2004) isolated strains related to Paenibacillus validus (a species that we have 
found repeatedly in unheated Antarctic soils) and a strain they identified as P apiarius 
on Mt Melbourne, and found that these organisms, from iron-rich soils, often bene- 
fited from iron supplements in their growth media (Pepi et al. 2005). The novel 
species B. luciferensis, B. shackletonii (Logan et al. 2002, 2004a), Paenibacillus 
cineris, and P. cookii were isolated from Candlemas Island; P. cookii, like 
B. fumarioli, has also been isolated from a gelatin production plant (Logan et al. 
2004b). The presence of Paenibacillus species in these soils is of particular interest, 
as these organisms often fix nitrogen; Rodriguez-Diaz et al. (2005) demonstrated 
the presence of the nifH gene in Paenibacillus wynnii, from unheated soil on 
Alexander Island, and capacity for acetylene reduction in P cineris and P. cookii. 
Logan et al. (2000) also found strains of the mesophiles B. sphaericus (also found 
on Alexander Island by Rodriguez-Diaz et al. (2005) ) and B. cereus, and the ther- 
mophile B. (now Geobacillus) thermodenitrificans on Candlemas Island. 



8.5 Adaptations for Growth at High Temperatures 

Organisms growing at high temperatures need enzyme adaptations to give molecular 
stability as well as structural flexibility, heat- stable protein- synthesizing machinery, 
and adaptations of membrane phospholipid composition. They differ from their mes- 
ophilic counterparts in the fatty acid and polar headgroup compositions of their 
phospholipids. The effect of temperature on the membrane composition of G. stearo- 
thermophilus has been intensively studied. Phosphatidyl glycerol (PG) and cardioli- 
pin (CL) comprise about 90% of the phospholipids, but as the growth temperature 
rises the PG content increases at the expense of the CL content. The acyl-chain com- 
position of all the membrane lipids also changes; the longer, saturated-linear and iso 
fatty acids with relatively high melting points increase in abundance, and anteiso fatty 
acids and unsaturated components with lower melting points decrease. As a result, the 
organism is able to maintain nearly constant membrane fluidity across its whole 
growth temperature range; this has been termed homeoviscous adaptation. An alter- 
native theory, homeophasic adaptation, considers that maintenance of the liquid- 
crystalline phase is more important than an absolute value of membrane fluidity in 
bacteria (Tolner et al. 1997). 

The major cellular fatty acid components of Geobacillus species following incu- 
bation at 55 °C are (with ranges as percent of total given in parentheses) iso-C 15 . 
(20-40%; mean 29%), iso-C 16 . (6-39%; mean 25%) and iso-C 170 (7-37%; mean 
19.5%), that account for 60-80% of the total (Nazina et al. 2001). The figures given 
by Fortina et al. (2001) for G. caldoxylosilyticus and Sung et al. (2002) for G. toebii 
generally lie within these ranges, with the exception that strains of the former species 
showed 45-57% of iso-C 150 . Such higher levels of iso-C 15 . are also found in 
Anoxybacillus species (Dulger et al. 2004). Geobacillus thermoleovorans subsp. 
stromboliensis (Romano et al. 2005), isolated from Italian geothermal soil, showed 



8 Aerobic, Endospore-Forming Bacteria from Antarctic Geothermal Soils 169 

fatty acid patterns within the ranges seen for other Geobacillus species. 
Thermobacillus xylanilyticus Touzel et al. (2000) shows a fatty acid profile 
dominated by iso C 16 . , whereas the profile of Vulcanibacillus modesticaldus 
is dominated by iso C 15 . (L'Haridon et al. 2006). Direct comparison of profiles 
between the obligately thermophilic species and mesophilic aerobic endospore 
formers is not normally possible, as the assays of members of the two groups have 
not usually been done at the same temperature. 

Nicolaus et al. (1995) studied the effects of growth temperature on polar lipid 
patterns of aerobic endospore formers from geothermal and unheated soils from 
Antarctica; at 60°C, the strain subsequently proposed as Bacillus thermantarcticus 
(and which presently awaits reassignment to Geobacillus) showed a level (27%) of 
iso-C 170 fatty acid which is similar to that of Bacillus thermoglucosidasius, but a 
high level (36%) of anteiso C 17 . fatty acid in comparison with Bacillus and 
Geobacillus species. Nicolaus et al. (2002) reviewed their lipid studies on Antarctic 
isolates. Strains tentatively identified as Bacillus showed increased phosphoglycolipid 
contents with increased growth temperature, at the expense of phosphoaminolipid and 
phospholipids; higher-melting point acyl chains such as iso-C 170 were favoured at 
maximum growth temperatures, whereas iso-C 15 . was synthesized at minimum 
growth temperature. 

Llarch et al. (1997) compared the fatty acid profiles of aerobic endospore form- 
ers isolated from Antarctic geothermal environments; their six isolates had temper- 
ature ranges with minima between 17 and 45 °C and maxima between 62 and 73 °C, 
with optima of 60 to 70°C. Two strains (temperature ranges 37-70 and 45-73 °C) 
were found to lie nearest to G. stearothermophilus in a phenotypic analysis, and 
two other isolates could be identified as strains of B. licheniformis (temperature 
range 17-68°C) and B. megaterium (temperature range 17-63°C) whose maximum 
growth temperatures were extended beyond those seen in strains from temperate 
environments. The fatty acid profiles for all of these strains were compared follow- 
ing incubation at 45 °C, and the results suggested that any potential distinctions 
between the rather variable fatty acid profiles of Geobacillus species and Bacillus 
species are largely lost when strains of each group are incubated at the same 
temperature. 

Members of Alicyclobacillus possess an apparently unique phenotype, as the 
main membranous lipid components of most species are co-alicyclic (co-cyclohexane 
or co-cycloheptane) fatty acids; it has been shown that co-alicyclohexyl fatty acids 
pack densely, resulting in low diffusion at high temperatures (Kannenberg et al. 
1984). Polar lipids based upon hopanoids are also important chemotaxonomic 
markers for this genus, and hopanoid content is increased in response to elevated 
temperatures at low pH. Together with lipids containing co-cyclohexane fatty acids, 
hopanoids are important for forming a biological membrane stable enough to with- 
stand extreme temperature and pH conditions. 

Nicolaus et al. (2002) found that the percentage of cyclohexyl fatty acids 
increased as the growth temperature was raised for their Antarctic Alicyclobacillus 
isolates. However, an Alicyclobacillus isolate from fruit juice, that did not possess 
co-alicyclic fatty acids, showed a fatty acid profile similar to that of Bacillus tusciae 
(Goto et al. 2003). 



170 N.A. Logan and R.N. Allan 

8.6 Nutrition and Growth Conditions of Thermophiles 

Most aerobic endospore formers are chemo-organotrophs and, despite the very 
wide diversity of the genus, will grow well on routine media such as nutrient agar 
or trypticase soy agar. However, some isolates, particularly those from nutritionally 
poor environments, may grow poorly if at all on these standard media because of 
their neutral pH, and/or insufficient salinity, or because they are nutritionally too 
rich. Most species will use glucose and/or other fermentable carbohydrates as sole 
sources of carbon and energy. Inorganic and organic sources of nitrogen are used. Many 
species will utilise an ammonium salt as their sole nitrogen source; amino acids are 
also widely utilized, and strains of some species can use urea. Most thermophilic 
species conform to this general nutritional pattern for aerobic endospore formers. 

Geobacillus species utilize a wide range of substrates, including carbohydrates, 
organic acids, peptone, tryptone, and yeast extract; the ability to utilize hydrocar- 
bons as carbon and energy sources is a widely distributed property in the genus 
(Nazina et al. 2001; Marchant et al. 2006). A strain of G. thermoleovorans has been 
found to have extracellular lipase activity and high growth rates on lipid substrates 
such as olive oil, soybean oil, mineral oil, tributyrin, triolein, and Tweens 20 and 40 
(Lee et al. 1999). 

Alley clobacillus species are obligately acidophilic, and have been found particu- 
larly in fruit juices and solfataric environments; isolates from the latter show differ- 
ent patterns of resistance to metal salts (Simbahan et al. 2004). 

Bacillus schlegelll grows chemolithoautotrophically, using H 2 as electron donor 
and C0 2 as carbon source (CO will satisfy both requirements), or chemo-organo- 
heterotrophically; it can also grow autotrophically on thiosulfate (Hudson et al. 
1988). Hydrogenase is constitutive and has a temperature optimum between 70 and 
75 °C. Carbohydrates are not used. Organic acids and a small number of amino acids 
are utilized as sole carbon sources, whereas ammonium ions, asparagine, and urea can 
be utilized as sole nitrogen sources. Bacillus tusclae also grows chemolitho- 
autotrophically, using H 2 as electron donor and C0 2 as carbon source, or chemo- 
organoheterotrophically. Carbon and nitrogen sources are similar to B. schlegelll, 
and some alcohols can also be used as carbon sources. 

Members of the genus Sulfobaclllus are facultative autotrophs that can obtain 
their energy by oxidizing ferrous iron, as well as elemental sulfur or its reduced 
compounds. None of the three established species from this genus have been 
reported from natural geothermal environments, but unidentified Sulfobaclllus 
strains have been reported from geothermal waters (see Table 8.1). 

Amino acid transport in G. stearothermophllus is Na + -dependent, which is unusual 
for neutrophilic terrestrial organisms, but common among marine bacteria and alka- 
lophiles; however, the possession of primary and secondary Na + - transport systems 
may be advantageous to the organism by allowing energy conversion via Na + - 
cycling. Although the phospholipid adaptations needed to give optimal membrane 
fluidity at the organism's growth temperature also result in increased proton perme- 
ability, this may be counteracted by increased proton pumping activity using the less 
permeable sodium ions as coupling ions (de Vrij et al. 1990; Tolner et al. 1997). 



8 Aerobic, Endospore-Forming Bacteria from Antarctic Geothermal Soils 171 

Allan (2006) used a [ 14 C] 1-glutamic acid tracing technique to study the type 
strains of Br. levickii and An. terranovensis with a view to understanding why they 
could not be isolated from the same habitats on Mt Melbourne and Mt Rittmann (see 
Section 8.4 above and Fig. 8.1). Results for both species showed distinct differences 
in the mechanisms used for 1-glutamic acid uptake; Br. levickii possesses a secondary 
uptake system specific for 1-glutamic acid which is dependent on K + and possibly 
H + , and An. terranovensis possesses multiple uptake systems which are capable of 
transporting other amino acids as well as 1-glutamic acid, a process which appears 
to be dependent on multiple factors including Na + , K + , H + , the electrical gradient 
across the cell membrane, and osmotic conditions. These studies showed that both 
strains utilize glutamate, which is probably available from cyanobacteria and micro- 
algae in their natural habitats (Siebert and Hirsch 1988). Glutamate is nonessential, 
however, as the organisms grew on a defined medium from which it had been omit- 
ted. For comparative purposes, 1-glutamic acid uptake by strains of B. fumarioli and 
B. cereus were also investigated. B. fumarioli was found to possess an uptake system 
similar to that of An. terranovensis (consistent, perhaps, with their similar habitats), 
whereas the B. cereus strain possessed multiple uptake systems capable of transport- 
ing both the d and 1 isomers of glutamic acid. 



8.7 Conclusions 

It is clear that our understanding of the bacteriology of Antarctic geothermal soils 
remains in its infancy. Although several new species of aerobic endospore formers 
have been discovered in these niches in the last 15 years, almost nothing is known 
about the relationships of the prokaryotes and eukaryotes within these environ- 
ments, whereas knowledge of the ecologies of deep-sea hydrothermal vents is ever 
increasing (Reysenbach et al. 2006). 

Acknowledgements We thank P. De Vos and colleagues at the University of Gent, Belgium, 
N. R. Russell, and BAS (especially the late D. D. Wynn- Williams, and P. Convey), and the Italian 
(especially R. Bargagli and B. Nicolaus) and Spanish Antarctic research programmes for helping 
us to make our contribution. 



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Chapter 9 

Peatland Microbiology 

Shwet Kamal and Ajit Varma(S) 



9.1 Introduction 

Peat contains a high proportion of dead organic matter, mainly plants, accumulated 
over thousands of years. Peatlands cover about 5-8% of the world's surface. 
Peatlands are important wetland areas maintaining supplies of clean water to rivers 
and acting as a carbon sink. Peatlands may contain 3-3.5 times the amount of carbon 
stored in tropical rain forests and act as an ancient habitat containing many rare and 
threatened species. As such, they are an unparalleled record of our past. 



9.2 General Description of Peatlands 
9.2.1 What Is Peat? 

Peat is a brownish-black material that is formed in acidic, anaerobic wetland (peat- 
land) conditions. It consists mainly of partially decomposed, loosely compacted 
organic matter with more than 50% carbon. It is made up of Sphagnum mosses, 
stems and roots of sedges and reeds, animal remains, plant remains, fruits, and 
pollens. Unlike most other ecosystems, the dead plants/animals in peatlands remain 
without decomposing for hundreds or thousands of years. This is because of water- 
logged conditions, where the lack of oxygen prevents micro-organisms from rapidly 
decomposing the dead plants/animals. As a result, the organic matter in peatland is 
easily identifiable. The formation of peat is a very slow process, and it takes 
approximately 10 years for 1 cm of peat to form. 



Ajit Varma 

Amity Institute of Microbial Sciences, Amity University Uttar Pradesh, Noida-201303 (India) 

e-mail : aj itvarma @ aihmr. amity.edu 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 111 

© Springer- Verlag Berlin Heidelberg 2008 



178 S. Kamal and A. Varma 

9.2.2 Description ofPeatlands 

A peatland is an ecosystem where the production rate of biomass exceeds its 
decomposition rate. The result is the accumulation of organic matter coming from 
plant debris, animal remains, and microbes. This more or less decomposed biomass 
forms the peat. Peatlands can also be defined as anaerobic and acidic wetlands 
containing peat. 

In a natural peatland ecosystem, the water table level is near the soil surface, 
which creates anaerobic conditions lowering the microbial activity. Peatlands 
are distinguished from marshes and swamps by the lower decomposition/ 
production ratio. 



9.2.3 Types ofPeatlands 

9.2.3.1 Bogs (Ombrotrophic Peatlands) 

Ombro trophic peatlands are fed only by rainwater or snow falling on their surface. 
The groundwater has no contact with the wetland and no nutrients come through 
water supplies from adjacent ecosystems. It is characterized by mineral deficiency 
and acidic conditions (pH 4.2) due to a dominant vegetation of Sphagnum. Because 
of their high ion exchange capacity and release of organic acids, Sphagnum species 
can lower the pH of their environment. The process of bog formation is called as 
ombrotrophication. 

Bogs have a diplotelmic soil structure, characterized by the presence of an 
acrotelm and a catotelm (Ingram, 1978). The catotelm is the bottom layer of 
peat that is permanently below the water table resulting in anaerobic conditions, 
low microbial activity, and peat decomposition. The acrotelm (30-50 cm), over- 
lying the catotelm, is characterized by periodically alternating anaerobic and 
aerobic conditions, as determined by water table fluctuations. This alternation 
accelerates microbial activity. The acrotelm has a loose structure comprising 
living parts of mosses, as well as dead and poorly decomposed plant material. 
It can contain and release large quantities of water that limit variations of the 
water table in peat bogs. 



9.2.3.2 Fens (Minerotrophic Peatlands) 

Fens are peatlands fed by precipitation (rainwater) as well as surface run-off water 
and are rich in basic cations due to contact with run-off water from mineral soil. For 
this reason fens are also called minerotrophic peatlands. The trophic status of fens 
varies between oligotrophic (poor) to eutrophic (rich), depending upon the concen- 
tration of basic cations (such as Ca, Mg, and Na). The pH of water and substrate of 
oligotrophic fen varies from 3.8 to 6.5, whereas for eutrophic fens it varies from 



9 Peatland Microbiology 179 

pH 5.8 to 8.4 (Sjors 1950). The vegetation of poor fens consists of Sphagnum 
mosses, whereas on rich fens herbaceous species of Cyperaceae family and brown 
mosses from Amblystegiaceae family are dominant. Dwarf shrubs and trees are 
also present on both poor and rich fens. 



9.2.4 Geographical Distribution ofPeatlands 

Peatlands cover almost 5-8% (500 million hectares) of the world's surface. The 
occurrence, extent, and types of peatland depend on the prevailing climate. Most of 
the temperate and boreal regions of the Northern Hemisphere offer favorable condi- 
tions for peatland development. 

The largest peatland areas of Asia are found in Indonesia, China, and Malaysia, 
covering a total area of 24.4 million ha, most of which occurs in the tropical zone 
of Indonesia. Tropical peatlands are most diverse, comprising fresh water swamp, 
peat swamps, as well as eutrophic, mesotrophic, and oligotrophic types. 

European peatlands are most extensive in Finland, Sweden, and Norway. Much 
of the European peat resources are threatened due to development. Natural peat- 
lands in the Netherlands are lost, Switzerland and Germany each have only 500 ha 
remaining, whereas the United Kingdom has seen a loss of >90% of bog peatland. 
Ireland has only 18% of its original peatland area left. 

North America has almost 40% of the total world's peatlands with 173 million ha. 
The main peatland areas of Canada are found in Manitoba, Ontario, Alberta, and 
Saskatchewan and are the least threatened peatlands. Over half of the United States 
peatlands occur in nearly undisturbed settings in Alaska. In South America peat- 
land occurs in Brazil, the Falklands, Mexico, Chile, Guyana, and Cuba. 

Peatlands in Australia and New Zealand occur in temperate zones, covering an 
area of 2.4 million ha. The bogs are found in Patagonia, Tasmania, New Zealand, and 
the Eastern Australia highlands. The most characteristic bog type in these regions is 
that of the cushion-plant bog, which does not occur elsewhere in the world. 

African peatlands are classified into five types: blanket bogs, raised bogs, valley 
peatlands, fens, and reed swamps, covering an area of 5.8 million ha. The major 
peatland areas are in the highlands of Burundi, Rwanda, South Africa, southwest- 
ern Uganda, the highland area of Zaire, and the Aberdane Range and the Cherangani 
Hills in western Kenya. 

The peatlands of Siberia cover a vast area, with 103 million ha existing in western 
Siberia alone. 



9.2.5 Peatland Development 

Peatlands formed originally either by filling in of shallow water bodies by peat and 
organic matter, or they form in areas where river flood plains, forests, grasslands, 
or rocky areas previously existed. These changes occur because of tectonic processes, 
local climatic conditions, and geology resulting in the formation of peatlands. 



180 S . Kamal and A. Varma 

Peatlands can develop by two processes: 

(a) Terrestrialization or infilling of shallow lakes: Terrestrialization occurs when a 
peatland is initiated from a lake or a water body 

(b) Paludification of poorly drained land: Paludification is a process where Sphagnum 
mosses invade and begin to accumulate peat on uplands. 

In the tropics, paludification is a common process of peatland formation 
(Gorham et al. 2003), whereas in temperate regions, terrestrialization serves as the 
primary mechanism of peatlands initiation followed by paludification (Anderson 
et al. 2003). Indeed, the water level influences peatland formation and develop- 
ment, but allogenic (external influences such as atmospheric humidity and tempera- 
ture) and autogenic factors (internal influences such as succession of vegetation, 
topography, substrate, and hydrology) also play an important role. A positive hydric 
regime (water input higher than water output), low temperature, and high precipita- 
tion favor paludification, whereas for terrestrialization allogenic factors can be in a 
wider range because the water table is already at the soil surface (Payette 2001) and 
even in drought periods the initiation of peatlands by terrestrialization is possible. 



9.2.6 Chemical Composition of Tropical and Temperate Peatland 

9.2.6.1 Organic Composition 

The chemical composition of peat materials is predominantly influenced by the 
parent vegetation, the degree of decomposition, and the original chemical environ- 
ment. Peat materials can be grouped into five fractions: 

(a) Water-soluble compounds 

(b) Ether and alcohol- soluble materials 

(c) Cellulose and hemicellulose 

(d) Lignin and lignin-derived substances 

(e) Nitrogenous materials or crude proteins 

The content of water-soluble compounds, mainly polysaccharides, mono-sugars, 
and some tannins, generally varies depending on the stage of decomposition. The 
ether and alcohol extracts contain fatty acids, waxlike components, resins, and 
nitrogenous fats and some waxes, tannins, various pigments, alkaloids, and soluble 
carbohydrates. The amounts are strongly related to the original vegetation. For 
example, Sphagnum peats may contain as much as 15% of soluble carbohydrates, 
reeds and sedge peats less than 5%. The cellulose and hemicellulose fraction 
decomposes easily and the content in the original vegetation is therefore usually 
greater than that in the derived peat. The lignin and lignin-derived materials com- 
monly constitute the largest portion of the peat because of poor decomposition 
(range 20-50%). Finally, the nitrogenous constituents are small with respect to 



9 Peatland Microbiology 181 

other fractions and mostly proteinaceous in nature. Total nitrogen may vary from 
0.3-4.0% (of dry weight). 



9.2.6.2 Elemental Composition 

There is generally a wide variation in mineral composition between different 
peats, but the principal constituents other than carbon, hydrogen, nitrogen, and 
oxygen are either silicon or calcium. The silicon usually comes from wind-blown 
minerals or washed-in sediments and is therefore of low abundance. The calcium 
content can be high in eutrophic environments. Together with magnesium, this 
element in its ionic form is strongly adsorbed onto the colloidal organic particles. 
Contents of iron, aluminium, sodium, and sulfur reach high levels in some peats 
due to environmental conditions. It is not clear whether tropical peats have 
elemental compositions essentially different from temperate peats. It is sug- 
gested, however, that because tropical soils commonly have larger sesquioxide 
contents than temperate regions, the tropical peat- forming environments and the 
peats themselves might generally reflect such a difference by higher iron and 
aluminium contents. 



9.2.7 Significance ofPeatlands 

Functions and values of peatlands make them valuable ecosystems. Their role as 
a carbon sink has gained visibility recently because of its impact on the green- 
house effect and climatic changes. Natural peatland emits greenhouse gases such 
as methane (CH 4 ), but they also stock a large amount of carbon present in plant 
debris and peat. 

Peat bogs also play a role in regulating water flow by stocking water in cases of 
abundant precipitation and releasing it during dry periods. Peat and peatland vege- 
tation both are extremely good water filters, making peatlands highly effective in 
removing sediments, pollutants, and pathogens. 

The minerals present in the peatlands contribute to the removal of the pollutants 
through chemical reactions such as denitrification and sulfate reduction. Some of the 
reactions require energy inputs, which are drawn from the large amount of carbon 
stored in the peatlands. Presence of peatlands downstream of industries and mines 
ensures filtering out and temporary storage of pollutants, including waste matter such 
as uranium from gold mining operations, thus protecting river systems. 

Due to slow decomposition rate and prevailing anoxic conditions, plant parts, 
especially seeds and pollen, are preserved in peat for thousands of years and thus 
these ecosystems act as paleoarchives. With modern dating techniques, it is possi- 
ble to reconstruct past environment and climate through the identification of seeds 
and pollen present within the superposed peat layers. 



182 S. Kamal and A. Varma 

Biodiversity is another criterion on which base peatlands deserve special status. 
Because they are unique acidic ecosystems, peat bogs support specific plant com- 
munities. A number of plant and bird species are found only in peatlands. 



9.2.8 Peatland Vegetation 

As with any other ecosystem, peatland vegetation depends upon the availability of 
nutrients in the soil, groundwater source, and base rock type. Plants that formed the 
earlier layers of peat hundreds of years ago may not be identical to present vegeta- 
tion of the peatland. The vegetation includes sedges, reeds, bulrushes, swamp forest 
trees, herbs, orchids, shrubs, and ferns. 

Peatland ecosystems represent a harsh environment for plants because of acidic 
and nutrient-poor conditions, a high water table, and exposition to desiccation due 
to the absence of protection against wind and sun, limiting the growth and survival 
of many plant species. A few plant communities such as Sphagnum mosses are the 
dominant vegetation, maintaining a water-logged condition due to their ability to 
retain water. They cause acidification by releasing humic acids and are efficient at 
absorbing and keeping nutrients. 

The shrubs include small-leaved cranberry, rhodora, Labrador tea, and leather 
leaf. These plants can tolerate very wet to very dry conditions, wind, and ice, and 
produce a toxin that prevents browsing by mammals. 

Three types of carnivorous plants are found in peatlands: pitcher plants, sundews, 
and bladderworts. Although these plants still photosynthesize, their carnivorous habits 
allow them to grow more vigorously in a competitive environment. The diet of 
carnivorous plants, which includes not only insects, but also mites, spiders, and even 
small frogs, has evolved to supply nitrogen which is lacking in the soil. The plant's 
prey, attracted by the red-veined leaves, is trapped by downward pointing bristles 
which not only prevent escape, but force it further down into the rain and dew held 
by the "pitcher." It is then digested by acid and proteolytic enzymes secreted by diges- 
tive glands of the plants and absorbed into the plant as food. In some cases certain 
bacteria are also helpful in digestion of the insect inside the plant. 

Trees often show unusual growth forms in peatlands, particularly in open areas 
of bogs where peat layers are often thickest. Orchids, with their elegant stalks and 
showy flowers, contrast with the thick-leaved shrubs and spiky sedges. 



9.3 Cycles in Peatlands 

9.3.1 Carbon Cycle in Peatlands 

Peatlands exchange carbon dioxide with the atmosphere. Carbon dioxide is seques- 
tered by vegetation by way of photosynthesis. Carbon is then accumulated as a peat 
deposit in the anaerobic catotelm. Carbon dioxide is released in the atmosphere 



9 Peatland Microbiology 



183 



through plant and microbial respiration; the two components together are referred to 
as "total respiration". Plants release C0 2 through mitochondrial respiration and pho- 
torespiration. Soil also emits C0 2 following the aerobic decomposition of organic 
matter (Clymo et al. 1998) and methane (CH 4 ) oxidation (methanotrophy) by bacteria 
(Sundh et al. 1995), two processes that occur in the acrotelm. However, the conditions 
in peatland limit decomposition, so most of the carbon is retained and stored in the 
peat. Figure 9.1 presents an overview of the carbon cycle in peatlands. 



Atmosphere 







C0 2 


CH 4 










j, 














1 




















Vegetation 


Photosynthesis 




Photo respiration 
























c 
o 

'w 

i 

b 


c 
o 

£ 

5 


c 
o 

'55 

i 

a 

) 






Acrotelm 




Aerobic 
decomposition 


















DOC 
leaching 




CH 4 

oxidation 
{methanotrophy 




3 




















Utter production 




A 


C 


H 4 
^ullition 








" Root exudation 




) 








1 \ 

Organic carbon ^ 

i 






/ 




CH4 via , 
plant transport / 


! 






Peat accumulation 








j 


k # 




Catotelm 






X* 




° < 


*<~ 


\ Ar 

de 
<m 


aerobic 
composition 




5 < 






ethan< 


>ge 


nests) 





Mineral soil 



■ Groundwater 



Fig. 9.1 The carbon cycle in a natural peatland. Methane production from peatlands, exchange of 
carbon dioxide with the atmosphere, and dissolved organic carbon (doc) processes are pre- 
sented. (Used with permission, Faubert (2004).) 



184 S. Kamal and A. Varma 

9.3.2 Cycle of Dissolved Organic Carbon in Peatlands 

Organic material is also released from the peatland as dissolved organic carbon 
(DOC; see Fig. 9.1), which gives a characteristic brown color to the peatland water. 
DOC is released by leaching and enters into downstream aquatic ecosystems 
(Dalva and Moore 1991). DOC exports from temperate and boreal peatlands range 
between 4 and 20 g nr 2 yr 1 (Moore 2001). Billett et al. (2006) investigated the 
spatial and temporal connectivity between concentrations of organic carbon in 
stream water and the soil carbon pool in a small (130 ha) upland catchment in 
northeastern Scotland by comparing downstream changes in dissolved organic 
carbon with spatial changes in the soil carbon pool and concluded that the linkage 
between stream water DOC concentrations and the soil carbon pool in the upper 
1.5 km of the stream are likely to be driven by temperature-related DOC production 
in near- surface peatlands. 



9.3.3 Methane Cycle 

Methane is produced by anaerobic decomposition of the organic matter (Cao et al. 
1996) in the catotelm by the process called methanogenesis. Plants stimulate 
methanogenesis by releasing "young" organic material as root exudates in the 
catotelm. This fresh carbon is easily decomposable by methanogenic bacteria forming 
methane, which is released in the atmosphere through three processes: diffusion, 
ebullition, and plant transport. Diffusion of CH 4 occurs throughout the peat column 
when this substrate is not oxidized by methanotrophic bacteria. Ebullition of CH 4 
happens via bubbles that are released to the atmosphere from water- saturated peat 
(Rosenberry et al. 2003) whereas vascular plants transport CH 4 to the atmosphere 
via the aerenchymatic tissues from the roots that are in the catotelm (Thomas et al. 
1996). These components of the CH 4 cycle (presented in Fig. 9.1) are controlled by 
temperature (Dunfield et al. 1993), water table position (Moore and Dalva 1993), 
peat chemistry (Svensson and Sundh 1992), and plant community factors such as 
bryophyte distribution (Bubier et al. 1995). Peatlands contribute almost 3 to 7% 
of the global annual emission of greenhouse gas methane. 



9.4 Diversity of Organisms Colonizing Peatlands 

The microflora in peatland soil consists of prokaryotes (bacteria and Archaea) and 
fungi (saprophytes and mycorrhizae). A soil fauna comprising nematodes, mites, 
colembola, and other insects is also present. All of these organisms play important 
roles in carbon cycling and interact with plants via exchange of organic and 
inorganic compounds. They also alter the physicochemical environment of the 
interacting organisms. 



9 Peatland Microbiology 1 85 

Direct counts of bacteria using epifluorescence microscopy and radioisotopic 
measurements of microbial degradative processes indicated bacterial densities of 
about 10 8 cells ml -1 of peatland water, irrespective of depth. Radioisotopic most- 
probable-number (MPN) counts of heterotrophs able to mineralize 14 C-labeled 
substrates to 14 C0 2 showed significant populations of glucose degraders (10 4 -10 6 
cells ml -1 ) as well as degraders of benzoate (10 2 — 10 3 cells ml -1 ), 2,4-dichlorophe- 
noxy acetate (10 2 -10 5 cells ml -1 ) and Sphagnum material (10 3 -10 7 cells ml -1 ) in the 
various peatlands examined. The MPN counts of N0 3 " reducers varied from 10 3 -10 6 
cells ml -1 , S0 4 " reducers from 10 2 -10 3 cells ml -1 , methanogenic bacteria from 
10 3 -10 6 cells ml -1 , and methane oxidizers (methanotrophs) from 10 3 -10 4 cells ml -1 , 
depending on sampling site and depth. Many pure cultures of aerobic bacteria and 
fungi have been isolated from various peatlands. Most of them can grow on organic 
compounds (carbohydrates, aromatic molecules, hydrocarbons, etc.) as sources of 
carbon. Some Bacillus bacteria are able to fix atmospheric N 2 . 



9.4.1 Insects 

Peatlands contain distinctive insects in addition to widely distributed generalists, 
including species restricted to bogs (tyrphobionts) and species characteristic of bogs 
but not confined to them (tyrphophiles). Bogs raised above the water table form 
characteristic habitat islands in southern boreal and temperate forest zones. The 
historical development and nature of individual bogs are reflected by differences 
among their insects, which are of great biogeographical and ecological interest. The 
environmental sensitivity of bogs also makes insects valuable as bioindicators. Some 
of the important peatland insects are the water beetles (whirling beetles and great 
diving beetles), water bugs (pond skaters), water boatmans, and water scorpions. 
They usually feed upon small flying insects and small animals. 



9.4.2 Fungi 

9.4.2.1 Mushrooms and Ectomycorrhizae 

Mushrooms typically seen in peatlands are dominated by saprotrophs and species 
of Galerina are particularly common. Another common species is the Yellowleg 
Bonnet, Mycena epipterygia. It is, however, very easy to identify as it has a thin 
jelly like layer on the cap which can be peeled off. Waxcaps are sometimes called 
the orchids of the fungal world and some small bright red species can be found in 
bogs. These are likely to be the Vermilion Waxcap, Hygrocybe miniata, and the 
Goblet Waxcap, Hygrocybe cantharellus or Hygrocybe helobia. Other fungi that 
can be found are species in the groups Stropharia (often on dung) and Hypholoma. 
There are the oddities such as the Bog Beacon, Mitrula paludosa and Sarcoleotia 



186 



S. Kamal and A. Varma 



turficola, which demonstrate the variety of forms of fungi. Any lone birch tree or 
clumps of birch around the bog edge will host a different suite of fungi with leaf 
litter decayers, species such as the Birch Polypore, Piptoporus betulinus, or the 
Birch Woodwart, Hypoxylon multiforme on the trees, or various ectomycorrhizal 
fungi around their base. The latter group help the tree to take up nutrients from the 
soil and protect it against soil pathogens in exchange for carbon that the tree produces 
by photosynthesis. Typical ectomycorrhizal fungi include the Tawny Grisette, 
Amanita fulv a, the Birch Milkcap, Lactarius tabidus, the Ghost Bolete, Leccinum 
holopus, the Birch Brittlegill, Russula betularum, and the Yellow Swamp Brittlegill, 
Russula claroflava. 

Some mycorrhizal type associations have evolved to help the woody plants to 
survive in low nutrient conditions, where shrubs and conifers co-exist in an organic 
matrix dominated by Sphagnum mosses. Ectomycorrhizal fungi produce an elaborate 
hyphal network, thus vastly expanding the root exploration zone (Fig. 9.2). 




Fig. 9.2 Extensive network of mycorrhizal hyphae radiating from roots of a larch (larix) seedling 
grown in peat. (Used with permission http://www.biology.ed.ac.uk/research/groups/jdeacon/ 
mrhizas/ecbmycor.htm#top) 



9 Peatland Microbiology 



187 



9.4.2.2 Orchid Mycorrhizae 

Orchid mycorrhizae are a symbiotic relationship between the roots of plants of the 
family Orchidaceae and a variety of fungi. All orchids are my cohetero trophic at 
some point in their life cycle. Orchid mycorrhiza activity is critically important 
during orchid germination, as an orchid seed has virtually no energy reserve and 
obtains its carbon from the fungal symbiont. Many adult orchids retain their fungal 
symbionts, although the respective benefits to the adult photo synthetic orchid and 
the fungus remain largely unexplored (Fig. 9.3). The fungi that form orchid mycor- 
rhiza are typically basidiomycetes. These fungi come from a range of taxa including 
Ceratobasidium (Rhizoctonia), Sebacina, Tulasnella, and Russula. Some orchids 
associate with saprotrophic or pathogenic fungi, whereas other orchids associate 
with ectomycorrhizal fungal species. These latter associations are often called tri- 
partite associations as they involve the orchid, the ectomycorrhizal fungus, and the 
ectomycorrhizal host plant. 



9.4.2.3 Ericoid Mycorrhizae 

Ericoid mycorrhizae are a symbiotic relationship between fungi and the roots of 
plants from the order Ericales. Ericoid mycorrhiza is considered crucial for the 
success of the family Ericaceae in a variety of edaphically stressful environments 
worldwide. Ericaceous plants commonly co-occur in soils with leguminous or 




Fig. 9.3 Orchid mycorrhiza, showing coils of fungal hyphae in cells of the protocorm. (Used with 
permission: http://www.biology.ed.ac.Uk/research/groups/jdeacon/mrhizas/ecbmycor.htm#top) 



188 S. Kamal and A. Varma 

carnivorous plants, further highlighting the low nutrient status of these soils. 
Ericoid mycorrhizal fungi enable their host plants to obtain nutrients in these poor 
soils. Ericoid mycorrhizas are formed between ascomycetous, and more rarely 
hyphomycetous fungi, and species of the Ericaceae and Epacridaceae. Members of 
the Ericaceae exhibit three types of mycorrhiza: ericoid, arbutoid, and monotro- 
poid. The ericoid type is the most important and is found in some genera as 
Calluna, Erica, Rhododendron, and Vaccinium. 

Ericoid mycorrhizas have evolved in association with plants that often dominate 
some of the most climatically and edaphically stressed environments. These plants 
characteristically become dominant when levels of acidity become extreme. Under 
these very low pH conditions, mineralization of nutrients is inhibited and metallic 
elements show maximum solubility. There is evidence that species of Calluna and 
Vaccinium have a very high constitutional tolerance to some stresses. Usually, the 
formation of ericoid mycorrhizae increases this tolerance and there is good reason 
to believe that their presence is essential for survival of the plant partner (Bhatnagar 
and Varma 2006). 

Generally, fungi forming ericoid mycorrhizae are from order Helotiales of the 
ascomycetes. These fungi include Rhizoscyphus ericae, Hymenoscyphus ericae, 
Oidiodendron maius, Myxotrichum arcticum, Phialocephala fortinii, and 
Leptodontidium orchidicola. 



9.4.3 Methanogens 

Methanogens are Archaea (Woese 1981) that occupy narrow ecological niches in 
peatland and other environments (see Chapter 7 for a discussion of methanogens in 
permafrost). They mediate the formation of methane from simple substrates (e.g., 
H 2 -C0 2 , formate, methanol, and acetate) in highly reducing and anaerobic environ- 
ments (Garcia et al. 2000). They have the most stringent requirements of all anaerobes 
for the absence of oxygen (<2ppm) and for growth they require a redox potential 
of less than -330 mV. 

Peatlands are wetland ecosystems where productivity exceeds biodegradation 
and except for the surface water layer, peats are anaerobic environments. As such, 
they represent suitable habitats for methanogenic bacteria. Williams and Crawford 
(1984) found a high population of 10 6 methanogen cells ml -1 of peatland interstitial 
water. The methanogenic food chain is a microbial system that mediates the bio- 
degradation of organic compounds in many anaerobic environments. The carbon 
flow through this chain avoids a buildup of inhibitory metabolic end products. 
Because methanogenesis is the terminal step in this anaerobic food chain, any per- 
turbation of the chain should be reflected by altered methane production. 
Consequently, methanogenesis is a key process to study, reflecting the combined 
activities of many different microbial groups. Over 65 species of methanogens 
belonging to 20 different genera are known today (Sowers, 1995). 



9 Peatland Microbiology 189 

In peatlands, H 2 and C0 2 -dependent methanogenesis is thought to be the main 
pathway for CH 4 production, but in some minerotrophic peatland (fens) acetoclastic 
methanogenesis is often predominant in upper peat layers (Popp and Chanton, 1999). 
The diversity of methanogenic communities of fen and bogs (Basiliko et al. 2003) 
has recently been described, but data on methanogenic pathways and methanogen 
populations are scarce. Galand et al. (2005) studied the methanogenic pathways in 
three peatland systems (i.e., mesotrophic fens, oligotrophic fens, and ombrotrophic 
bogs) and found that mesotrophic fens harbour the lowest production rates of CH 4 
from H 2 and C0 2 . Because H 2 -C0 2 and acetate are the main precursors of CH 4 , the 
fraction of CH 4 not produced by H 2 -C0 2 is predominantly the result of acetoclastic 
methanogenesis. 

Methanogenesis occurs in three steps. Decomposition of organic matter in 
anaerobic environments first occurs by hydrolysis, performed by bacteria. This is 
followed by fermentation, performed by bacteria and some Archaea, and methano- 
genesis, performed exclusively by members of the domain Archaea. Methanogens 
produce methane by the reduction of carbon dioxide using hydrogen as an electron 
donor, or through the cleaving of acetate into methane and carbon dioxide 
(Luton et al. 2002). Sauer and Thauer (2000) showed that methyl-coenzyme 
M (2-methylthioethane sulfonate) is the key intermediate of methane formation in 
a methanogenic Archaea {Methanosarcina barkeri). It is generated from coenzyme 
M (5-2-mercaptoethane sulfonate) in methyl transfer reactions catalyzed by 
proteins containing zinc. 

The methanogens of peatland ecosystems mainly belong to five orders. 
Members of the Methanomicrobiales are found in bog peatlands only, whereas 
Methanosarcinales, Methanococcales, and Methanopyrales are found in fen peat- 
land. Methanobacteriales are generally found in bogs, but some genera of 
Methanobacteriales occur in fens. Methanogenium organophilium and Methano- 
spirillum hungatei are the most frequently isolated genera of Methanomicrobiales 
whereas Methanobacterium bryantii is a common member of the Methanobacteriales. 
Methanosarcina mazei, Methanosarcina barkeri, Methanosarcina sicilliae, 
Methanosarcina acetivorans, and Methanosaeta spp. are the important organisms 
identified from the order Methanosarcinales, whereas Methanococcus jannaschii 
and Methanococcus infernus are peatland colonizers of the order Methanococcales. 
The order Methanopyrales comprises Methanopyrus kandleri. Although all these 
organisms are involved in methanogenesis, their mode of action differs. The 
Methanomicrobiales and Methanobacteriales probably account for hydrogeno- 
trophic methanogenesis and Methanosarcinales for acetoclastic methanogenesis. 
A pathway for the generation of methane by a representative of the 
Methanomicrobiales is presented in Fig. 9.4. 



9.4.3.1 Acidophilic Methanogens 

Until recently, it was widely accepted that methanogens require neutral to slightly 
alkaline pH conditions (pH 6.8 to 8.5) for optimum growth and methane production. 



190 



S. Kamal and A. Varma 



O -C- O Caioon dioxide 
Formylmethanofuran 




Formylmtthanofuran 
foimyitratisferast 





Formylittelhanofuraji 



Formyl - H^MPT 



Methenyl - H4MPT 



Fco indep ende nt Methenyl - HjMPT Methenyl - H^ 
dehydrogenase ^dehydrogenase 

t^XIjO ob ** ^ - ?E 

LJL^ - y^Y^k^^, f -<^^* om ft Methylene -HjMPT 

I Methylene - H<MFT <_ 
^reductase k— 1 *^ 

V^"tt^ I Methyl -H<MPT lfr jL^ 



oMethyl-I^MPT 



Methyltransferase 



,^v 



Methyl coenzyme M 



, 



Methyl coenzyme M 
rnethylredurtase 



Methane 



H 

H 

Fig. 9.4 Flow diagram of methane generation from carbon dioxide and hydrogen by 
Methanobacterium thermoautotrophicum. In an initial reaction of the pathway, formylmethano- 
furan dehydrogenase catalyses the reductive conversion of carbon dioxide and methanofuran to 
formylmethanofuran, with the required electrons being provided by molecular dihydrogen. (Used 
with permission: http://umbbd.msi.umn.edu/meth/meth_image_map.html; Ellis et al. 2006) 



Attempts were made to isolate acid-tolerant methanogens and a methanogen iden- 
tifed as Methanobacterium uliginosum has been reported to grow at pH 6.0 to 8.0 
(Konig 1984). Also, Methanobacterium espanolae was found to grow at pH 5.5 to 
6.2, which can be considered as an example of a truly acidophilic methanogen. 



9 Peatland Microbiology 191 

Certain strains of Methanosarcina have been shown to grow at low pH, using 
methanol and H 2 as the substrate (Maestrojuan and Boone 1991). 



9.4.3.2 Psychrophilic Methanogens 

The Archaea that are most readily isolated from naturally cold environments and 
that are most amenable to laboratory cultivation are methanogens. Isolates have 
come from an Antarctic lake (Franzmann et al. 1997), a freshwater lake in 
Switzerland (Simankova et al. 2001), and cold marine sediment in Alaska (Chong 
et al. 2002) and the Baltic Sea (Singh et al. 2005). In addition, various Methano- 
sarcina, Methano spirillum, Methanocorpusculum, and Methanomethylovorans 
species have been isolated from a Boreal fen, tundra, a polluted pond in Russia, lake 
sediment in Switzerland, and manure digested at low temperature in a laboratory. 
Methanococcoides burtonii and Methanogenium frigidum were isolated from Ace 
Lake, Antarctica (Franzmann et al. 1997). M. frigidum uses H 2 and C0 2 for growth 
and is nonmotile; M. burtonii uses methyl substrates (trimethylamine and methanol) 
and is flagellated and motile. As M. burtonii is a methylotrophic methanogen, it 
does not compete with hydrogen-utilizing, sulfate-reducing bacteria in the environ- 
ment. M. burtonii is cosmopolitan in cold environments, with a close relative 
{Methanococcoides alaskense; 99.8% 16S rRNA identity) isolated from Skan Bay, 
Alaska (Singh et al. 2005) and closely related strains identified from an Antarctic 
bay (Purdy et al. 2003; 99% 16S rRNA identity) and deep-sea sediment (Li et al. 
1999; 98.8% 16S rRNA identity). 

Although no individual micro-organism can grow at both the freezing and boiling 
points of water, methanogens are thermally diverse and there are species that can 
grow at 110°C (Methanopyrus kandleri), 0°C (M. burtonii), and all temperatures 
in between (Saunders et al. 2003). This illustrates that methanogenesis and the 
main energy and biosynthetic pathways are not restricted by growth temperature; 
that is, novel pathways and cellular processes are not essential for cold (or heat) 
adaptation. 



9.4.3.3 Factors Affecting Methanogenesis 

Methanogenesis is highly dependent on environmental conditions such as temperature, 
water table, content of organic matter, pH, and so on (Segers 1998). Temperature is 
known to exert a significant influence on methanogenesis and the number of metha- 
nogens in lake sediments. In culture, methanogenic bacteria metabolize best in the 
pH range of 6.7 to 8.0. However, very low rates of methanogenesis have been 
observed at pH 5.8 (Dedlysh et al. 1998; see also above, this section). Methanogens 
utilize sulfide or cysteine to satisfy their sulfur requirements; however, high sulfide 
concentrations have been shown to inhibit methanogenesis in sediments (Khan et al. 
1979). Phosphate also has been found to inhibit methanogenesis in lake sediments, 



192 S. Kamal and A. Varma 

as has nitrate (Balderston and Payne, 1976) in salt marsh sediments. Ammonium 
ion may slow the conversion of acetic acid to methane at high concentrations 
(Wolfe and Higgins, 1979). Methanogenic substrates such as hydrogen (Winfrey 
et al. 1977) and acetate (Cappenberg, 1974) often stimulate methanogenesis in lake 
sediments. Glucose stimulated methanogenesis in some habitats (Winfrey et al. 
1977), however, at glucose concentrations greater than 10 g l" 1 , suppression of 
methane production occurred. Vitamins and yeast extract (Wolfe and Higgins, 
1979) also have been observed to stimulate methanogenesis. Finally, sulfate has 
been found to inhibit methanogenesis in sediments (Winfrey et al. 1977). 



9.4.4 Methanotrophs 

Aerobic methanotrophic bacteria using methane as the carbon and energy source 
are an integral part of many natural peatland ecosystems where methane is pro- 
duced and consumed. Methanotrophs maintain a balance of atmospheric methane 
and play a crucial role in the global methane cycle. They oxidize methane through 
methanol and formaldehyde to carbon dioxide and incorporate carbon into biomass 
at the level of formaldehyde. They typically occur at the aerobic/anaerobic interface 
of wet environments, for example, lake sediments, rice paddies, tundra soils, and 
bogs (Sundh et al. 1994; Whalen et al. 1996). The methanotrophs use methane 
diffusing from the anaerobic zone and often operate as a biofilter for methane. In 
this way they reduce and control its potentially hazardous emission to the atmos- 
phere. Methane is one of the most effective greenhouse gases and contributes to as 
much as 20% of total global warming. 

Methanotrophs have been divided into three major categories: type I, type II, and 
type X, which differ in phylogeny, chemo taxonomy, internal membrane ultrastruc- 
ture, carbon assimilation pathways, and some other biochemical features (Bowman, 
2006). Currently, type-I methanotrophs, that belong to the family Methylococcaceae 
of the Gammaproteobacteria, include six validated genera, that is, Methylomonas, 
Methylobacter, Methylomicrobium, Methylosphaera, Methylosarcina, and Methylo- 
halobius. They possess lamellar stacks of intracytoplasmic membranes (ICM), 
assimilate formaldehyde produced from the oxidation of methane or methanol via 
the ribulose monophosphate (RuMP) pathway, and have DNA G + C content ranging 
from 45 to 55mol%. 

In contrast, type-II methanotrophs belong to the family Methylocystaceae of 
the Alphaproteobacteria and include four validated genera: M e thy lo sinus, 
Methylocystis, Methylocella, and Methylocapsa. They generally have ICM 
located on the cell periphery, a higher level of G + C content in their DNA, and 
assimilate formaldehyde via the serine pathway. 

The intermediate type-X methanotrophs belong to the Gammaproteobacteria and 
include Methylococcus and Methylocaldum genera, thermophilic and thermotolerant 
representatives of which possess ICM similar to those of type-I methanotrophs, but 
have a DNA G+C content of 55 to 65mol%. They express the RuMP cycle for 



9 Peatland Microbiology 193 

formaldehyde assimilation and enzymes of the serine pathway and the ribulose 
bisphosphate (RuBP) cycle. The traditionally recognized type-X category has now 
been incorporated into type I (Wise et al. 1999). 



9.4.4.1 Detection of Methanotrophs 

Many studies were conducted on the diversity and ecology of methane-oxidizing 
bacteria (MOB). Cultivation-based approaches have been shown to reflect a highly 
distorted picture of the original in situ community structure. Cultivation-independent, 
molecular approaches (mostly by cloning and sequencing, denaturing gradient gel 
electrophoresis (DGGE), and/or terminal restriction fragment length polymorphisms 
(T-RFLP)) have been based on the 16S rRNA gene, or on functional genes for the 
methane mono-oxygenases. Two types of such genes are known: pmoA, encoding the 
particulate methane mono-oxygenase (Lukow et al. 2000) and mmoX, encoding 
the soluble methane mono-oxygenase (Auman et al. 2000; see also below, this 
section). Yet another possibility is mxaF (McDonald and Murrell, 1997), encoding 
the methanol dehydrogenase, the next key enzyme in the pathway of methane 
metabolism. The most used marker genes are the pmoA and the 16S rRNA gene. 

The use of pmoA enables us to focus on MOB and functionally related bacteria 
as well as to detect and identify thus far uncultured MOB . The sequence phylogeny 
of pmoA corresponds closely to that of 16S rRNA, and pmoA has therefore been 
extensively used for the cultivation-independent identification of MOB from 
diverse environments. The sequence of pmoA, encoding the 27-kDa subunit of 
pMMO, reflects evolutionary relationships amongst pmoA containing bacteria 
(Nguyen et al. 1998). 

Studies on the diversity of MOB and the ecology of microbial methane oxidation 
were performed in aqueous environments (fresh, brackish, marine and underground 
hot spring water, and aquifers), sediments (marine and freshwater), mud, various 
soils (rice field, forest, arable, pasture, peat bog, landfill cover; arctic to tropical), 
deep-sea smokers, and shallow-sea methane seeps as well as the tissues of marine 
invertebrates. Soil is by far the most studied environment with respect to MOB 
ecology (Bourne et al. 2001). Studies have revealed a high diversity of MOB but 
the factors influencing competition between different MOB, determining their eco- 
logical niches and maintaining the high diversity observed, are largely unknown, 
except for atmospheric methane oxidation and competition between type-I and type-II 
MOB . The main factors in the competition between mesophilic neutrophilic type-I 
and type-II MOB (co-existing in many environments) are methane and oxygen partial 
pressures and the availability of fixed nitrogen. Type-I MOB appear to outcompete 
type-II strains under oxygen-rich, methane-limited conditions. Most type-II MOB 
and some type-I (particularly Methylomonas, Methylobacter, and Methylococcus) 
strains are capable of nitrogen fixation, thus having a competitive advantage 
under nitrogen-limited conditions. Some type-I MOB form desiccation-resistant 
or -sensitive cysts. Type-II MOB forms exospores conferring some resistance 
against fluctuations in nutrient supply. 



194 S. Kamal and A. Varma 

9.4.4.2 Psychrotrophic Methanotrophs 

Aerobic methanotrophic bacteria typically occur at the aerobic/anaerobic interface 
of wet environments, such as lake sediments, rice paddies, in particular land, tundra 
soils, and bogs (Sundh et al. 1994; Whalen et al. 1996). Tundra soils contain 15% 
of the biosphere's total organic carbon. Thus, the response of this large carbon 
reservoir to global climate warming could be important. There is increasing 
evidence that bacterial oxidation of methane is a vital regulator of CH 4 emission. 
Field and laboratory experiments on methane consumption revealed that tundra 
soils of Unalaska Island and the Aleutian Islands consumed methane at soil 
temperatures of 7°C and at CH 4 concentrations ranging from below to well above 
ambient levels (Whalen and Reeburgh 1990). These experiments also demonstrated 
that soil oxidation of atmospheric CH 4 (at a concentration of 1.7ppm) was microbe- 
mediated and indicated the presence of a soil population capable of oxidizing CH 4 
at concentrations tenfold lower than the ambient atmospheric concentrations. 

Because the kinetic properties of methanotrophs are not consistent with their 
growth under atmospheric CH 4 concentration, the occurrence of any novel methan- 
otroph having a cold active microbial methane oxidase with a high affinity for CH 4 
would be of great interest. Scanning electron microscopy of tundra bog soils 
revealed the presence of bacterial microcolonies on organic residues sampled from 
the Carex and Sphagnum layers. Bacteria absorbed on the particles gave positive 
reactions with fluorescent antibodies targeting Methylocystis parvus, Methylocystis 
trichosporium, Methylobacter bovis, and Methylobacter capsulatus (Trotsenko and 
Khmelenina 2005; Vecherskaya et al. 1993). In this study, the number of methano- 
trophs in peat layers ranged from 0.1 to 22.9 x 10 6 cells g" 1 of soil, thus comprising 
from 1 to 23% of the total bacterial population. The representatives of Methylomonas, 
Methylobacter, Methylococcus, Methylo sinus, and Methylocystis genera occurred 
simultaneously in all soil samples. Type-I methanotrophs were more abundant than 
type II and members of the genus Methylobacter were predominant. 

Micro-organisms residing in tundra soils face low temperatures, long periods in 
the frozen state, high water content due to poor drainage at times causing anaerobi- 
osis, acidic pH, and a narrow range of nutrients. Such extreme conditions might 
favor the existence of multistress-resistant methanotrophs, which differ from those 
in the temperate soil community. A pure culture of a psychrophilic methanotroph 
that grows between 3.5 and 20°C and optimally at 5 and 10°C was first isolated 
from tundra soil and identified as a new species, Methylobacter psychrophilus 
(Tourova et al. 1999). 



9.4.4.3 Acidophilic Methanotrophs 

A novel acidophilic methanotroph was described, Methylocella palustris. Strains of 
M. palustris were isolated from acidic Sphagnum peat bogs (Dedlysh et al. 1998) 
and classified as type-II methanotrophic bacteria. However, phylogenetically 
these acidophilic strains were only moderately related to the known type-II 



9 Peatland Microbiology 195 

methanotrophs and were more closely affiliated with the heterotrophic bacterium 
Beijerinckia indica subsp. indica. The isolation of M. palustris from Sphagnum 
bogs of different geographical locations (four different sites in west Siberia and 
European north Russia) suggests that these bacteria might be widely distributed in 
acidic wetlands of the northern hemisphere. However, information on the distribu- 
tion and abundance of Methylocella in northern wetlands is still lacking. As the 
result of their profound distinctness from other known methanotrophs, these 
organisms have not been targeted by the culture-independent 16S ribosomal DNA 
(rDNA)-based molecular approaches developed for detection of type-I and type-II 
methano trophic bacteria (Costello and Lidstrom 1999). 



9.4.4.4 Methane Oxidation 

Methane oxidation is divided into "low affinity" and "high affinity" categories. 
Typically, methanotrophs in culture oxidize high concentrations of methane (low- 
affinity methanotrophs), whereas in situ methane oxidation kinetics measurements 
indicate that there are high-affinity methanotrophs in soils. The distinction between 
these two populations is unclear because extant methanotrophs can also exhibit 
properties of high-affinity methanotrophs under certain conditions. The oxidation of 
methane at atmospheric levels (high affinity methane oxidation), mostly found in 
upland soils, appears to be mainly associated with the predominance of two uncul- 
tured MOB based on molecular analyses (Holmes et al. 1995; Pedersen 1996). 

The bacterial methane oxidation pathway is catalyzed by one of the two types of 
the enzyme methane monooxygenase (MMO). The soluble cytoplasmic MMO 
(sMMO) is found in only some of these bacteria whereas the particulate membrane- 
bound MMO (pMMO) is present in virtually all known MOB except, perhaps, the 
Methylocella species. The pMMO consists of three membrane-associated polypep- 
tides encoded by pmoC, pmoA, and pmoB (Pacheco-Oliver et al. 2002). In addition 
to pMMO, most type-II (Methylo sinus, Methylocystis) and some type-I methano- 
trophs (Methylomonas, Methylomicrobium, and Methylococcus) possess sMMO. 
The enzyme from the strains belonging to Methylosinus, Methylocystis, and 
Methylococcus has been thoroughly studied and the nucleotide sequence of the sMMO 
gene cluster mmoX, mmoY, mmoB, mmoZ, mmoC, and mmoD appears to be highly 
conserved. The pmoA gene, encoding a 26-kDa subunit that harbors the active site 
of the pMMO, and the mmoX gene, coding for the a subunit of the sMMO hydroxy- 
lase component, can be used as appropriate gene markers for the occurrence of the 
enzymes in various methanotrophs. 

Methanol dehydrogenase (MDH), the second enzyme involved in methane 
oxidation, is present in all gram-negative methylotrophs including methane and 
methanol utilizers. This enzyme oxidizes the methanol produced from the oxidation 
of methane by methane monooxygenase (MMO). The mxaF gene, encoding the 
large subunit of the enzyme, is an appropriate indicator gene for occurrence of 
methylotrophs in the environment (McDonald and Murrell, 1997). MDH is a 
pyrroloquinoline quinone-linked enzyme that carries out a key step in bacterial 



196 S. Kamal and A. Varma 

one-carbon (CI) metabolism because it catalyzes the oxidation of methanol to 
formaldehyde, the intermediate of both assimilative and dissimilative metabolism 
in methylotrophs. It is distinct from the alcohol dehydrogenase of gram-positive 
methylotrophic bacteria (de Vries et al. 1992) and methylotrophic yeasts (Williamson 
and Paquin, 1987). 

Analysis of the predicted amino acid sequences corresponding to various 
structural genes for MDH, or mxaF genes, carried by various methanotrophs and 
methylotrophs revealed strong sequence conservation (McDonald and Murrell, 
1997). Of the 172 amino acid residues, 47% were conserved among all 22 
sequences obtained in this study. Phylogenetic analysis of these mxaF sequences 
showed that those from type-I and type-II methanotrophs form two distinct clusters 
and are separate from mxaF sequences of other gram-negative methylotrophs. 
Sequences of mxaF retrieved by PCR from DNA isolated from a blanket bog peat 
core sample formed a distinct phylogenetic cluster within the mxaF sequences of 
type-II methanotrophs and may originate from a novel group of acidophilic 
methanotrophs. 



9.4.4.5 Factors Affecting Methanotrophs 

The methanotrophic bacteria are affected by environmental factors such as pH, 
temperature, salinity, and water table level. The activity increases as aeration of the 
acrotelm is enhanced after water level draws down. This affects CH 4 emissions, 
which are lower as CH 4 consumption is higher (Svensson and Sundh 1992). In 
some sites, a net consumption of CH 4 has been found (Martikainen et al. 1995). 
This situation may occur for peatlands of temperate regions, but not necessarily for 
peatlands of the boreal and subarctic regions. With the higher temperatures caused 
by global warming, these peatland types are expected to release more CH 4 because 
methanogenic bacteria will be favored by the melting of the permafrost. In that 
sense, CH 4 emission rates will be different according to the geographical location 
of peatlands (Moore et al. 1998). Ambient pH from 4.3 to 5.9 favors the growth of 
methanotrophs. The MOB are inhibited by acetylene, which acts as a suicidal sub- 
strate, being transformed by both forms of MMO into active intermediates that in 
turn readily react with cell compounds containing hydroxy 1 and amino groups. 



9.4.5 Antagonistic Bacteria 

Because of their antimicrobial activity, Sphagnum plants were used as a natural 
medicine in the old Indian and Maya cultures, and as wound dressing during the 
First and Second World Wars (Frahm, 2001). Although they are colonized by 
diverse bryophilous ascomycetes, no substantial fungal diseases of these plants are 
known (Dobbeler 1997). 

The screening of 493 bacterial isolates for antagonistic activity against fungal 
pathogens resulted in 237 (48%) active isolates (Opelt et al. 2007). The majority of 



9 Peatland Microbiology 197 

the antagonists belonged to the genera Serratia (15%), Burkholderia (13.5%), 
Staphylococcus (13.5%), and Pseudomonas (10%). Interestingly, a high proportion 
of antagonists, for example, Staphylococcus, Hafnia, Yersinia, and Pantoea, were 
identified as strains that are known as facultative pathogens of humans. Thus, 
Sphagnum plants represent an ecological niche not only for diverse and extraordi- 
nary microbial populations with a high potential for biological control of plant 
pathogens, but also for opportunistic human pathogens. 



9.4.6 Epiphytic Bacterial Communities 

Associations between algae and bacteria are commonly observed in peatlands. 
Culture and microscopy studies have documented a number of bacterial-algal inter- 
actions. Much attention has been focused on the release of dissolved organic carbon 
by algal cells and its support of bacterial growth, and the surfaces of living cells 
may also provide microenvironmental conditions favorable for bacterial processes 
that otherwise could not occur under ambient water conditions (Paerl and Pinckney, 
1996). Heavy bacterial colonization of algae is generally considered a sign of algal 
senescence, but colonization of young, active algal cells or colonies is also observed 
(Rosowski and Langenberg 1994) and benefits to algae of such associations have 
been frequently reported (Keshtacher-Liebson et al. 1995). Bacteria and algae may 
also compete for inorganic nutrients, and many algal taxa produce compounds that 
are potentially inhibitory to bacterial growth (Kellam and Walker, 1989). 

Epiphytic bacterial communities within the sheath material of three filamentous 
green algae, Desmidium grevillii, Hyalotheca dissiliens, and Spondylosium pul- 
chrum (class Charophyceae, order Zygnematales), collected from a Sphagnum bog, 
were characterized by PCR amplification, cloning, and sequencing of 16S ribos- 
omal DNA (Fisher et al. 1998). By phylogenetic analysis, the cloned sequences 
were placed into several major lineages of the bacterial domain: the Bacteroidetes 
phylum and the Alpha-, Beta-, and Gammaproteobacteria (Fisher et al. 1998). 

The representatives of the Bacteroidetes phylum mainly consisted of 
Sphingobacterium heparinum, S. thalpophilum, Flavobacterium mizutaii, Flavo- 
bacterium ferrugineum, Saprospira, Cytophaga arvensicola, Flexibacter filiformis, and 
Flexibacter sancti. Members of the Alphaproteobacteria included Rhodo spirillum 
rubrum, Rhodo spirillum fulvum, and Azospirillum lipoferum. Some other identified 
species in the Alphaproteobacteria were Methylobacterium, Beijerinckia indica, 
Rhodopseudomonas acidophila, and Methylosinus. Purple phototrophic bacteria 
belonging to the Betaproteobacteria were also observed and included Rubrivivax 
gelatinosus, Comamonas testosteronii, Rhodoferax fermentans, and Variovorax 
paradoxus. Hydro genoluteola thermophilus and Rhodocyclus, also of the 
Betaproteobacteria, were isolated. Acinetobacter Iwoffii was the only epiphytic 
species belonging to the Gammaproteobacteria. 

The bryophytes, particularly the mosses, are a diverse group of land plants that 
usually colonize habitats with moist or extremely variable conditions. One of their 
most important features is their life cycle, which involves alternation between a 



198 S. Kamal and A. Varma 

diploid sporophyte and a dominant, free-living haploid gametophyte generation. 
Very little is known about the interaction of bryophytes with bacteria. Bacterium- 
host interactions can be symbiotic, commensal, or pathogenic. Therefore, bacteria 
associated with three bryophyte species, Tortula ruralis, Aulacomnium palustre, 
and Sphagnum rubellum, which represent typical moss species of peatlands at the 
southern Baltic Sea coast in Germany, was analyzed using culture-dependent and 
culture-independent techniques (Opelt and Berg, 2004). 

A high proportion of uncultured or unidentified eubacteria were found from the 
surface of the mosses. Photorhabdus luminescens, Collimonas fungivorans, and 
diverse Pseudomonas spp. were identified. DNA analysis assigned two dominant 
bands obtained from Sphagnum to Pseudomonas grimontii and Methylobacterium 
mesophilicum. Species of Acetobacter, Frateuria, and Acidocella, bacterial genera 
known for their occurrence in acidic environments, were also found for Sphagnum. 
Tortula-specific bands were identified as Pseudomonas aeruginosa, Rhodococcus 
erythropolis, and Acidovorax wohlfahrtii. 



9.4.7 Microbial Use of Nitrogen and Phosphorus 

Peatlands contain up to 30% of the total organic nitrogen reserve of the world's 
soils, and thus have a potential to exert a significant influence on the global atmos- 
pheric budget of nitrous oxide (N 2 0; Martikainen et al. 1993). Microbial N 2 pro- 
duction, primarily through nitrification and denitrification processes, may account 
for a substantial fraction of the total N loss from the peatlands (Jacks et al. 1994), 
either directly as N 2 or, after further reduction, as dinitrogen (N 2 ) (Allen et al. 
1996). Large losses of N in gaseous forms are commonly related to waterlogged 
conditions combined with high N mineralization rates. Thus, N loss in the gaseous 
forms may be a significant mechanism for the removal of N from peatlands. 

Denitrification is the most important N 2 0-producing process in waterlogged 
peat soils (Velthof and Oenema, 1995), where oxygen is limited whereas nitrate 
and carbon are available for micro-organisms (Regina et al. 1996). Denitrification 
is an anaerobic microbiological process in which carbon serves as the energy 
source and nitrate as the electron acceptor, and mainly involves the bacterial genera 
Pseudomonas, Micrococcus, Bacillus, and Thiobacillus. In the process, nitrate is 
reduced to the gaseous nitrogen compounds N 2 and N 2 . N 2 can also be formed 
during nitrification, that is, oxidation of NH 4 + (Koops et al. 1997). A proportion of 
50% of water-filled pore space is the optimum for N 2 production in nitrification 
and up to 35% of the total gaseous nitrogen loss from agricultural land is through 
N 2 production by nitrification. N0 2 is an intermediate product in both nitrification 
and denitrification processes. The N0 2 resulting from nitrification activity can in 
turn be reduced to N 2 and N 2 and the term "nitrifier denitrification" has been 
coined to account for this particular mode of nitrogen loss from soils (Koops 
et al. 1997). 



9 Peatland Microbiology 199 

9.5 Conclusions 

Peatlands are important and unique systems for biogeochemical cycling of carbon, 
nitrogen, and other chemical elements. Because of the prevailing conditions in 
these waterlogged, mineral-poor, and biomass conservation-prone environments, 
metabolic processes occurring in peatlands represent particular variations of mech- 
anisms occurring in soils and water. As such, peatlands provide a specific contribution 
to the maintenance and changes in planetary biological systems. Global atmos- 
pheric and climatic changes will be influenced by peatland ecology. The future of 
peatlands bears direct relevance to the establishment of biospheric equilibria and, 
in this sense, the understanding of interactive processes occurring in these wetland 
systems, as well as measures for their preservation and rational use, bears direct 
relevance to humanity's future and the form this future will take. 

Acknowledgments The authors are thankful to CSIR, New Delhi for partial financial support. 



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Chapter 10 

Subsurface Geomicrobiology of the Iberian 

Pyritic Belt 



Ricardo Amils(K), David Fernandez-Remolar, Felipe Gomez, 

Elena Gonzalez-Toril, Nuria Rodriguez, Carlos Briones, 

Olga Prieto-Ballesteros, Jose Luis Sanz, Emiliano Diaz, Todd O. Stevens, 

Carol R. Stoker, the MARTE Team 

10.1 Introduction 

Terrestrial subsurface geomicrobiology is a matter of growing interest. On a funda- 
mental level, it seeks to determine whether life can be sustained in the absence of 
radiation, whereas it also aims to develop practical applications in environmental 
biotechnology. Subsurface ecosystems are also intriguing exobiological models, 
useful for the re-creation of life on early Earth (Widdel et al. 1993) or the represen- 
tation of life as it would occur in other planetary bodies (Boston et al. 1992). 
Subsurface ecosystems were originally reported in basalt aquifers (Stevens and 
McKinley 1995; Chapelle et al. 2002) and later in sedimentary aquifers, petroleum 
reservoirs, and alkaline and saline goldmine groundwater (Lin et al. 2006). Results 
obtained by deep-sea subsurface exploration initiatives are widening the scope of 
our knowledge in this field (D'Hondt et al. 2004). In this field there is a serious 
debate on whether the source of electron donors and/or acceptors is dependent on 
radiation-mediated reactions and also on contamination problems associated with 
drilling technologies, their mitigation, and control. In spite of the interest of subsur- 
face ecosystems, information concerning microbial abundance, diversity, and sus- 
tainability is still scarce, mainly due to methodological limitations. 

Among the different minerals, the metallic sulfides have the potential to be a 
good source of energy for subsurface chemolithotrophs. The micro-organisms that 
aerobically oxidize iron sulfides are well characterized (Gonzalez-Toril et al. 2003), 
however, little is known about the possibility of subsurface chemolithoautotrophic 
metabolism in anoxic conditions. The Mars Analog Research and Technology 
Experiment (MARTE) project (Stoker et al. 2004; Fernandez-Remolar et al. 2005a) 
outlined in this chapter was designed to search for this type of life in the nonporous 
volcanically hosted massive sulfide deposits (VHMS) of the Rio Tinto basement at 
Pena de Hierro (Iberian Pyritic Belt). 



Ricardo Amils 

Centro de Astrobiologia (INTA-CSIC), 28850 Torrejon de Ardoz, Madrid, Spain and 
Centro de Biologia Molecular (UAM-CSIC), Cantoblanco, 28049 Madrid, Spain 
e-mail: ramils@cbm.uam.es 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 205 

© Springer- Verlag Berlin Heidelberg 2008 



206 R. Amils et al. 

10. 2 The Rio Tinto Ecosystem 

Rio Tinto is an unusual ecosystem due to its size (100-km long), constant acidic pH 
(mean pH 2.3), high concentration of heavy metals (Fe, Cu, Zn, As, Mn, and Cr), 
and high level of microbial diversity, mainly eukaryotic (Lopez- Archilla et al. 
2001; Amaral-Zettler et al. 2002; Aguilera et al. 2006). 

Rio Tinto rises in Pena de Hierro, in the core of the Iberian Pyritic Belt (IPB), 
and reaches the Atlantic Ocean at Huelva. The IBP is one of the largest massive 
sulfidic deposits on Earth, formed as a hydrothermal deposit during the Paleozoic 
accretion of the Iberian Peninsula (Boulter 1996; Leistel et al. 1998). One important 
characteristic of Rio Tinto is the high concentration of ferric iron and sulfates 
present in its waters, products of the bio-oxidation of pyrite, the main mineral com- 
ponent of the IPB system. 

Pyrite, with its wide distribution on our planet, is considered an important 
substrate for chemolithotrophic metabolism because both of its components, sulfide 
and ferrous iron, can be used by sulfur- and iron-oxidizing micro-organisms as a 
source of energy. The first acidophilic strict chemolithotroph described, 
Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans), was isolated 
from an acidic pond in a coal mine more than 50 years ago (Colmer et al. 1950). 
Although At. ferrooxidans can obtain energy by oxidizing both reduced sulfur 
compounds and ferrous iron, much attention was paid in the past to the sulfur oxidation 
reactions due to bioenergetic considerations (Amils et al. 2004) 

The discovery that some strict acidophilic chemolithotrophs, such as 
Lepto spirillum spp. or Ferroplasma spp., could grow using ferrous iron as their 
only source of energy, and that these micro-organisms are mainly responsible for 
industrial metal bioleaching processes (biomining) and the generation of acid mine 
drainage (AMD), has changed this perspective, shifting the interest from sulfur to 
iron oxidation (Golovascheva et al. 1992; Edwards et al. 1999; Malki et al. 2006). 

The mechanisms by which acidophilic chemolithotrophs obtain energy by 
oxidizing metallic sulfides has remained controversial for many years (Ehrlich 
2002). But the recent demonstration that the ferric iron present in the cell wall and in 
the extracellular polysaccharides of acidophilic chemolithotrophic micro-organisms 
is responsible for the electron transfer from the mineral substrate to the electron 
transport chain has clarified this issue, with important fundamental and applied 
consequences (Gehrke et al. 1995; Sand et al. 1995, 2001). 

The differences observed during bioleaching of diverse metallic sulfides depend 
on the type of chemical oxidation promoted by ferric iron, which is determined 
by the crystallographic characteristics of the mineral. Under normal conditions of 
pressure and temperature, pyrite and two other sulfides, tungstenite and molybdenite, 
can only be oxidized by ferric iron through the so-called thiosulfate mechanism. 
Thiosulfate can then be further oxidized to sulfate, also by ferric iron (Sand et al. 
2001). The rest of the sulfides undergo oxidation through the poly sulfide mecha- 
nism. Polysulfide can be further oxidized to elemental sulfur. In this case the 
production of sulfate requires a subsequent biooxidation reaction promoted by 



10 Subsurface Geomicrobiology of the Iberian Pyritic Belt 207 

sulfur-oxidizing micro-organisms (e.g., At.ferrooxidans). These reactions are merely 
chemical oxidation reactions. The critical role of iron-oxidizing micro-organisms in 
these processes is to maintain a high concentration of the oxidant agent, ferric iron, 
by means of an aerobic respiration mechanism (reaction 1): 

Fe 2+ + Vi0 2 + 2H + -> Fe 3+ +H 2 

In addition, it is now well established that iron can be oxidized anaerobically 
in the absence of oxygen, when coupled to anoxygenic photosynthesis or to 
anaerobic respiration using nitrate as an electron acceptor (Widdel et al. 1993; 
Benz et al. 1998). 

As mentioned, the most important characteristic of Rio Tinto is the high con- 
centration of ferric iron and sulfates found in its waters. Ferric iron is responsible 
for the maintenance of the constant pH of the water due to its buffering potential 
(reaction 2): 

Fe 3+ + 3H 2 <h» Fe(OH) 3 + 3H + 



10.3 Geomicrobiology of the Iron Cycle 
in the Rio Tinto Ecosystem 

The combined use of conventional (enrichment cultures, isolation, and phenotypic 
characterization) and molecular microbial ecology methods (amplification of 
16-18S rRNA genes and its resolution using electrophoresis in denaturating 
conditions (PCR-DGGE), fluorescence in situ hybridization (FISH and CARD- 
FISH) and molecular cloning) has led to the identification of the most representative 
micro-organisms of the Rio Tinto basin (Gonzalez-Toril et al. 2003, 2006). Eighty 
percent of the diversity of the water column corresponds to micro-organisms 
belonging to only three bacterial genera: Lepto spirillum, Acidithiobacillus, and 
Acidiphilium, all members of the iron cycle (Gonzalez-Toril et al. 2003). All 
Leptospirillum isolates from Rio Tinto are aerobic iron oxidizers. At. ferrooxidans 
can oxidize iron aerobically and reduce it anaerobically (Malki et al. 2006). All 
Acidiphilium isolates can oxidize organic compounds using ferric iron as an electron 
acceptor and some isolates can do so in the presence of oxygen. Although other iron- 
oxidizing (Ferroplasma spp. and Thermoplasma acidophilum) or -reducing 
("Ferromicrobium" spp.) micro-organisms have been detected in the Tinto ecosystem 
(Gonzalez-Toril et al. 2003), their low numbers suggest that they play a minor role in 
the operation of the iron cycle, at least in the water column. 

Concerning the sulfur cycle, only At. ferrooxidans is found in significant num- 
bers in the water column of the Tinto ecosystem. This bacterium can oxidize both 
ferrous iron and reduced sulfur compounds. The oxidation of reduced sulfur 
compounds can be carried out aerobically and anaerobically. Some sulfate-reducing 



208 



R. Amils et al. 



Oxic 
|0 2 |* 



At. ferretoxidans 
AL fhfooxidans 
, cafdas 




Anoxic 

l< 



<CH 3 0) n 

Acidiphitium a pp. 
Aeidimterobium spp. 
^Ferromicrobium" spp. 



(CH 2 0)„C 




>4/. ferrovxidaas 
L*femx*xid<ms 
Ferroplasma spp. 



Fig. 10.1 Geomicrobiology of the Rio Tinto basin ecosystem associated with the iron and sulfur 
cycles. In blue are indicated the metabolic reactions associated with the iron cycle, and in red 
those associated with the sulfur cycle, operating in aerobic (pink) or anaerobic conditions (green). 
Identified microbial activities are associated with the different reactions operating in the Rio Tinto 
ecosystem. SRB: sulfate-reducing bacteria. The buffering capacity of ferric iron and 
the maturation of iron minerals have been placed in the model due to their relevance to the mass 
balance of the system 



activity has been detected associated with sediments in certain parts of the river 
(Malki et al. 2006). Figure 10.1 shows the integrated geomicrobiological model of 
Rio Tinto, in which the iron cycle plays a central role. 

As mentioned above, besides the extreme physicochemical conditions found in the 
Tinto ecosystem, what makes Rio Tinto a unique acidic environment is the unex- 
pected degree of eukaryotic diversity found in its waters (Lopez- Archilla et al. 2001; 
Amaral-Zettler et al. 2002; Aguilera et al. 2006). Members of the phylum Chlorophyta 
(Chlamydomonas, Chlorella, and Euglena) are the most frequent species followed by 
two filamentous algae belonging to the genera Klebsormidium and Zygnemopsis. The 
most acidic part of the river is inhabited by a eukaryotic community dominated by 
two species of the genera Dunaliella and Cyanidium, well known for their acidity 
and heavy-metal tolerance (Visviki and Rachlin 1993; Visviki and Santikul 2000). 
Among the eukaryotic decomposers, fungi are very abundant and exhibit great diver- 
sity, including yeast and filamentous forms (Lopez- Archilla et al. 2005). The mix- 
otrophic community is dominated by cercomonads and stramenopiles related to 
different genus (Bodo, Ochroomonas, Labyrinthula, and Cercomonas). The protistan 
consumer community is characterized by two species of ciliates, tentatively assigned 



10 Subsurface Geomicrobiology of the Iberian Pyritic Belt 209 

to the genera Oxytrichia and Euplotes. Amoebas related to the genera Valhkampfia 
and Naegleria can be found in the most acidic part of the river, and one species of 
heliozoan belonging to the genus Actinophyris seems to be the most characteristic 
predator of the benthic food chain of the river (Aguilera et al. 2006). 

Unicellular forms are not the only eukaryotes to develop in the extreme conditions 
of the Rio Tinto basin. Different plants can be found growing in the acidic soils of the 
riverbanks. The strategies used by these plants to overcome the physiological 
problems associated with the extreme conditions of the habitat are diverse. Some are 
resistant to the heavy metals concentrated in the soils in which they grow (Rodriguez 
et al. 2007; Berazain et al. 2007). Others specifically accumulate metals in different 
plant tissues. Recent analysis by X-ray diffraction (XRD) and Mossbauer spectros- 
copy of the iron minerals found in rhizomes and leaves of Imperata cylindrica, an 
iron hyperaccumulator perennial grass growing in the Rio Tinto banks, showed 
significant concentrations of jarosite and iron oxyhydroxides (Rodriguez et al. 2005). 
These results suggest that the management of heavy metals in general, and iron in 
particular, is much more complex and versatile in plants than has been reported to 
date (Schmidt 2003). Also, these results prove that multicellular complex systems can 
also develop in extreme conditions, like those existing in Rio Tinto. 

Most of the biomass of the Tinto ecosystem is located on the riverbed and 
the surface of the rocks, forming dense biofilms composed mainly of filamentous 
algae and fungi in which prokaryotic micro-organisms are trapped. Heterotrophic 
protists associated with these biofilms have been also detected (Aguilera et al. 2007). 
Significant iron mineral precipitation occurs on the surface of these negatively 
charged biofilms, generating iron bioformations, which grow with time following the 
hydrological cycles of the river. The age and depth of these iron formations strongly 
support the idea that Rio Tinto corresponds to a natural system and not to an 
industrially contaminated site (van Geen et al. 1997; Davis et al. 2000; Elbaz-Poulichet 
et al. 2001). It is obvious that mining activity during the last 5,000 years has altered the 
Tinto system (Avery 1974), but evidence of its antiquity can be found in massive lami- 
nated iron beds in three iron terraces occupying different elevations above the present 
river. The oldest of these, Alto de la Mesa, lies 60 m above the current river level. 
Preliminary isotopic data indicate an age of 2 million years (My) for this formation 
(Fernandez-Remolar et al. 2005b), although biostratigraphic considerations indicate 
that some altered minerals of the area (in situ gossan) may be as old as 6 My, suggest- 
ing that the IPB acidic water systems are of still older origin (Moreno et al. 2003). 

The combination of bioleaching processes and high evaporation rates induce the 
formation of concentrated brines in the origin section of the river (Fernandez- 
Remolar et al. 2003). Iron oxides associated with sulfates are the characteristic 
minerals that are formed in the modern sediments and young terraces: these are 
hydronium jarosite, schwermannite, copiapite, coquimbite, natronojarosite, gypsum, 
and other sulfate minerals. Goethite and hematite are the predominant minerals in 
the old terraces of the Tinto Basin (Fernandez-Remolar et al. 2005b). 

It is clear from these results that the main characteristics of Rio Tinto are not due 
to acid mine drainage of exposed mineral resulting from mining activity. Our working 
hypothesis predicts the existence of a continuous underground reactor in which the 



210 



R. Amils et al. 



sulfidic minerals of the IPB are the main energy source, and the river is the exhaust 
pipe that releases the products of the different metabolic reactions developing in the 
reactor. To test this hypothesis, we drilled a series of boreholes that intercepted 
groundwaters within the ore body in order to detect evidence of both subsurface 
microbial activity in the retrieved cores and potential resources to support these 
microbial communities in situ (MARTE project). 



10.4 Subsurface Geomicrobiology of the Iberian Pyritic Belt 

The main goal of the MARTE project, a collaborative effort between NASA and the 
Centro de Astrobiologia (INTA-CSIC, Spain), was the search for subsurface micro- 
bial activity associated with the IPB. The selected study site was Pena de Hierro 
(Figs. 10.2 and 10.3) on the north flank of the Rio Tinto Anticline, which com- 
prises a thick volcano sedimentary succession composed of dark shales, basaltic 
lavas, rhiolitic materials, fine ashes and tuffites, and green/purple shales. The 
hydrothermal activity is recorded as complex-massive sulfide lenses or stockwork 
veins of pyrite and quartz, which occur at the upper part of the IPB volcanic 
sequence (Leistel et al. 1998). 

The Pena de Hierro stratigraphy is inverted as a consequence of the Hercynian 
Orogenesis tectonism which produced an inverted anticline propagating along a 




Fig. 10.2 View of Pena de Hierro in which the MARTE project has taken place 



10 Subsurface Geomicrobiology of the Iberian Pyritic Belt 



211 



Borehole 4 



Borehole 8 




Hydrogeology 

• Neutral springs 
^ Ferric springs with 

pH above 3.5 

• Low pH feirin springs 
^ Low pH fen-pus springs 



Thrusting faults 
Normal faults 



B 



Geology legend 



Srite 
tfi 



pyrite 



Mine tailings 
Gossan 
Dark shales 
Purple shale 
Greenish ashes 

Acidic tuff (Host rock) 
feminized at base 
Pyroclasts, andesite 
■ s a nds , volcanic 
agglomerates and lavas 



50y 

5 My 

<340My 

i 

2 



Winning activity 

Acidic to neutral weathering 
Erosion of the Hercynian orogen 

Marine sediments 
Fine vol canociastic sedimentation 
In marginal environments 
Generation of andesite pyroclasts 
under acidic explosive eruptions 

Poorly transported andesite deposits 
alternating with lava deposits 



Post-Quaternary 

Neogene 
Upper-Vtsean 

=■» 

1] 



Fig. 10.3 Geological, hydrological, and stratigraphic maps of the Pena de Hierro field site. The 
location of boreholes and springs sampled are shown together with the stratigraphic sequence and 
ages of the geological units 



110°N-thrusting front. This compressive structure is intersected by NNE-SSW 
normal fractures that are related to the generation of acidic streams in the Pena 
de Hierro area and which are considered the origin of the Rio Tinto (Fig. 10.3). The 
reversed succession is topped by a gossan unit originated by the Tertiary in situ 
weathering of the sulfide complex. 

Despite the low porosity of these rocks, the use of complementary techniques 
including field survey, transient electromagnetic sounding, and drilling, showed 
that fractures play an essential role in storing groundwater. Faults intersect the 
Early Carboniferous volcanic tuff-hosted pyrite bodies, which are the primary 
energy source for the chemolithotrophic micro-organisms found in the Rio Tinto 
basin. The analysis of samples from drill cores, core leachates, and borehole fluids 
have shown distinctive microbial activity, appearing at different depths depending 
on environment variables in the aquifer. 



212 R. Amils et al. 

10.4.1 Drilling and Sample Analysis 

Drilling sites were selected using surface and subsurface techniques, including 
geological mapping, and surface hydrogeological and geophysical surveys 
(Jernsletten 2005). The well locations were selected to monitor spatial changes in 
microbial and hydrogeochemical processes. Coring was carried out using a 
commercial coring rig at three locations designated wells BH1, BH4, and BH8 (Fig. 
10.3). The boreholes were continuously cored by rotary diamond-bit drilling using a 
Boart-Longyear (Salt Lake City, UT, USA) HQ wireline system that produced 60- 
mm diameter cores within a plastic liner. Water was used as drilling fluid to refrig- 
erate the bit. Chemical tracers (NaBr) were used for controlling contamination 
introduced by the drilling fluids. Upon retrieval from the drill rig, cores were 
divided into 1-m lengths, flushed with N 2 , sealed, and transported to a nearby 
laboratory for geomicrobiological analysis. 

Samples were prepared aseptically in anaerobic conditions using an anaerobic 
chamber and subjected to different biological and geological analysis. Culture- 
independent detection of micro-organisms was done by epifluorescent microscopy 
after staining samples with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) 
and by fluorescence in situ hybridization with specific probes (FISH and CARD- 
FISH) (Gonzalez-Toril et al. 2006). Also a protocol has been developed for microbial 
biodiversity identification on subsurface cores. It is based on the amplification and 
molecular cloning (Sambrook and Russell 2001) of the metagenomic DNA extracted 
from the powdered samples (1-10 g), followed by the sequencing of the 16/18S 
rDNA genes of a representative number of molecular clones per sample (between 10 
and 20) and, when possible, the taxonomic assignment of the clonal sequences by 
means of molecular phylogenetic analyses. 

Chemolithotrophic enrichment cultures were performed in a minimal Mackintosh 
medium, with the addition of ferrous iron (20 g l" 1 ) or of a sterile rock sample, as 
electron donor (Gonzalez-Toril et al. 2006). Anaerobic enrichments were performed 
as described previously, for denitrifying micro-organisms with the addition of 
lOg l- 1 Na 2 S 2 3 and 1 g L 1 KN0 3 (Stevens and McKinley 1995), for sulfate reducers 
according to Gonzalez-Toril et al. (2006), and for methanogens according to Sanz 
et al. (1997). 

After drilling, the wells were completed by installing PVC casings set in clean 
gravel packing. Underground sampling for water and gas aquifer analyses was done 
by the installation of multilevel diffusion samplers (MLDS) at different depth 
intervals. These were inserted in perforated sections of the PVC casings and allowed 
to equilibrate for various periods of time (between three and twelve months). 

Rock leachates were produced by adding sterile anoxic water to powdered core 
subsample and allowing them to stand for 1 hour in anaerobic conditions before 
filtration and analysis. Water samples from the MLDS were filtered and the pH 
adjusted by adding HC1 to a 0.5 N final concentration. Anion concentrations were 
determined by ion chromatography using a Dionex (Sunnyvale, CA, USA) 4010i 
system with an AS14A column, and metal concentrations were determined by ion 



10 Subsurface Geomicrobiology of the Iberian Pyritic Belt 213 

chromatography using a CS5A column. Dissolved gases were sampled by allowing 
them to equilibrate across a submerged sealed polyethylene tube. Tubes were 
removed and analyzed within 1 hour by gas chromatography using a Carle Gas 
Chromatograph with a molecular sieve column and thermal conductivity detector, 
using purified N 2 as a carrier gas. 



10.4.2 Results from 2003 and 2004 MARTE Drilling Campaigns 

The characterization of the groundwater entering the ore body at Pena de Hierro 
was done by analyzing springs upslope. The water from these springs was aerobic, 
with a pH near 6 and a low ionic strength. The environment within the ore body was 
sampled by drilling boreholes BH4 (2003 drilling campaign) and BH8 (2004). 
Wells BH4 and BH8 cored around 165 m of pyrite stockwork. The lithology of 
borehole BH4 is shown in Fig. 10.4. The water table was encountered at nearly 
90 m below the surface. From top to bottom, the lithology of borehole BH4 consists 
of ca. 10 m of hydrothermally altered and weathered tuff, ca. 20 m of gossanized 
ores composed of goethite and hematite, ca. 120 m of coarse-grained volcanogenetic 
tuff of ryolithic composition that contains the sulfide ore body, and ca. 10 m of 
chloritized volcanic tuff with disseminated pyrite and carbonate traces. The sulfide 
ore was a complex mixture of polymetallic sulfide minerals dominated by pyrite 
(Fernandez-Remolar et al. 2008). 

Rock leachate analyses were performed to detect contamination by drilling fluids 
(tracers) but also to estimate resources available to micro-organisms from the solid 
phase. Sulfate was abundant and is a good indicator of the degree of oxidation of 
the ore. Surprisingly, nitrite and nitrate were present at concentrations higher than 
lOOppm in many samples. The analysis of the drilling fluid gave very low concen- 
trations of sulfate and nitrate, and there was no correlation between bromine and 
anion concentration, so the presence of these anions in the core samples cannot be 
due to surface contamination. Both Fe(II) (average concentration 95ppm) and 
Fe(III) (average concentration 22ppm) could be leached from powdered ore 
samples after a 1 hour incubation with 0.5 N HC1 or HC1 with hydroxylamine, 
which is a standard method for measurement of iron availability (Chao and Zhou 
1983; Lovley and Phillips 1987). Organic carbon content of the cores was near the 
detection limit of 0.01%. From the rock leachate experiments, we can conclude that 
electron acceptors for anaerobic respiration, particularly Fe(III), S0 4 2 ", N0 2 ", N0 3 ", 
and carbonates, are available from the volcanically hosted massive sulfide deposits 
(VHMS) rock matrix. 

Borehole fluids from the MLDS experiments were analyzed as a proxy for 
formation fluids, which could not be extracted from these low-porosity rocks. 
Formation water in BH4 was sampled with the MLDS from 85-105 and from 
135-150 mbls (meters below surface), several months after drilling. The measured 
composite pH was ca. 3.5, and has remained acidic for the two sampling years after 
drilling. Bromide in some samples suggested that these contained between and 



214 




R. Amils et al 

ThtoButfBtes DAP1[RS} Iron Nplrgn MahanaQena LAL 



Fig. 10.4 Geomicrobiological observation within the pyrite ore body. Core lithology and loca- 
tions of biological indicators for BH4. Blue shaded area at left indicates water table. Columns left 
to right: 1, example images of cores from each lithology; 2, lithology; 3, growth of denitrifying 
thiosulfate-oxidizing organisms in anaerobic chemolithotrophic enrichment cultures ; 4, detection 
of micro-organisms by fluorescence microscopy; 5, growth of iron-oxidizing organisms in aerobic 
chemolithotrophic enrichment cultures with ferrous iron; 6, growth of organisms in aerobic 
chemolithotrophic enrichment cultures with rock samples as source of energy; 7, growth of 
methanogens in enrichment cultures with added H 2 ; 8, positive limulus amebocyte lysate (LAL) 
assay. Solid lines in colums 3-8 indicate positive results in samples without detectable bromine 
tracer; empty lines correspond to instances with detectable bromide, indicating that some drilling 
fluid is present in the sample 



2% of residual drilling fluid. However, this level of contamination is too low to 
account for the solute concentrations that were detected in the BH4 MLDS 
formation water. Dissolved iron ranged from 108 to 480 ppm with an average of 



10 Subsurface Geomicrobiology of the Iberian Pyritic Belt 



215 





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Fig. 10.5 Concentration (in ppm) of solutes in BH4 formation water sampled by MLDS. Solutes 
include bromide (a), sulfate (b), iron (II) and (III) (c), and the dissolved gases H 2 (d), CH 4 (e), 2 
(f), and NO (g). For the dissolved gases, equilibrium concentrations are given 



188 ppm (Fig. 10.5). The dissolved ferric to ferrous ratio ranged from 0.3 to 4.3 
with an average of 1 and did not appear to correlate with total dissolved iron 
concentration. Sulfate concentration was relatively constant except near the water 
table, and was ca. 1,000-fold lower than in rock leachates. Neither nitrate nor nitrite 
were detected in the water. Small quantities of oxygen and N0 2 gas were present in 
some samples, and the two were inversely correlated. Dissolved methane was 
detected in many of the MLDS samples, indicating that methanogenic activity 
occurred throughout the ore body (Fig. 10.5). 

Dissolved hydrogen concentrations averaged 25 ppm, except in a zone within the 
massive pyrites, just below the water table, from 90 to lOOmbls, where concentrations 



216 R. Amils et al. 

ranged from 100 to l,000ppm (Fig. 10.5). A similar pattern was observed in the sec- 
ond borehole, BH8, with an average H 2 concentration measured 12 months after 
drilling of ca. 25 ppm, and with isolated zones of higher concentration. 

Electron donors available in the VHMS for microbial metabolism included 
Fe(II) and reduced S, which was expected, but also H 2 . Laboratory experiments 
showed that H 2 could be produced, in concentrations similar to those in most 
MLDS samples, by reaction of VHMS rocks with water. Potential mechanisms for 
this have not yet been investigated (Wachtershauser 1988; Drobner et al. 1990; 
Apps and van de Kamp 1993). We hypothesize that H 2 production supports metha- 
nogenesis throughout the wet sections of the VHMS. 

Aseptically collected rock core samples, in which no tracer could be detected, 
were used to test for the presence or absence of micro-organisms, and to estimate 
their distribution. However, sampling artifacts may arise while using this protocol, 
inasmuch as fractured zones, which are most likely to contain microbial habitats, are 
also likely to be invaded by drilling fluids, thus being excluded from further analysis. 
It is likely that the tracer system used to monitor contamination was conservative, 
because many "contaminated" samples used as controls appeared to be sterile. 

Micro-organisms were detected in different uncontaminated samples using culture- 
dependent and culture-independent methods. Distribution of micro-organisms was 
heterogeneous along the column, as expected in a system dominated by fracture 
flow. Using enrichment cultures, aerobic chemolithoautotrophs, mainly pyrite and 
iron oxidizers, and anaerobic thiosulfate oxidizers using nitrate as electron acceptor, 
sulfate reducers and methanogens were enriched from several samples. 

DAPI stain and fluorescence in situ hybridization (FISH) with probes of different 
specificities did not yield good results due to the high level of epifluorescence 
exhibited by the mineral components present in the samples. The use of catalyzed 
reported deposition modification (CARD-FISH) improved enormously the contrast 
between the hybridization signal and the mineral substrate. Using this technique, 
we have been able to unambiguously prove the presence of active micro-organisms in 
different uncontaminated samples and to show that these micro-organisms occurred 
at extremely low density in these samples (Fig. 10.6). Those low densities revealed 
by in situ hybridization could explain in part the difficulties to grow them in the 
enrichment cultures. Higher numbers could be seen in samples from cracks (Fig. 
10.7), which were normally discarded due to the presence of bromide, a signal of 
possible contamination by the drilling fluid. 

A protocol for a direct extraction of DNA from uncontaminated samples has been 
developed. Preliminary results showed that amplifiable and clonable DNA could be 
extracted from the rock samples, leading to the detection of bacteria related to the 
Acidithiobacillus genus and sulfate-reducing bacteria in the anaerobic section of 
wells BH4 and BH8, together with different heterotrophic bacteria which had been 
enriched under strict anaerobic conditions in both wells. The combination of enrich- 
ment cultures with in situ hybridization and direct cloning- sequencing experiments 
should allow us to identify the subsurface microbial diversity of the IPB and to 
develop geomicrobiological models of the different cycles operating in the system. 



10 Subsurface Geomicrobiology of the Iberian Pyritic Belt 



217 




Fig. 10.6 Fluorescence in situ hybridization (CARD-FISH) of a core sample. Core sample 8,50a 
retrieved from an uncontaminated section hybridized with the specific probe for Bacteria, EUB 388 




Fig. 10.7 Scanning electron microscopy of BH4 core sample 8,66a (155mbls) 



218 R. Amilsetal. 

The environment down-gradient from the ore body was sampled by drilling 
borehole BH1. We considered that in this zone, fluids would represent the end product 
of subsurface interaction with the VHMS. Well BH1 cored 59 m of the younger dark 
shales. Core samples from BH1 consisted of carboniferous greenish shales derived 
from volcanic ash with fine sandy lenses and lutites bearing organic matter, and were 
overlaid by 7 m of mine tailings. From 7 to 12.5mbls, shales were pale from ongoing 
leaching by groundwater. From 12.5 to 46m, shales were unweathered. Below 46 m, 
shales were altered to a fine noncohesive black material, suggesting aqueous weather- 
ing under anoxic conditions (Fernandez-Remolar et al. 2008). 

As expected, sulfate and iron concentrations were lower in leachates from BH1 
shales than in those from BH4 pyrites. Only small amounts of N0 3 " were detected 
in the leachates. Oxygen was not detected within the aquifer zone. Where present, 
dissolved sulfate in groundwater was in much higher concentrations than in 
groundwater from BH4, indicating that these waters had experienced more interac- 
tion with the ore. Neither N0 2 ~ nor N0 3 " were detected in water samples; however, 
dissolved NO x gases were present at concentrations slightly higher than in water 
samples from BH4. Dissolved H 2 , where detected, was at concentrations lower than 
in BH4 but still sufficient to make H 2 available as a microbial electron donor. 
Methane concentrations were several orders of magnitude higher than at BH4. 
These observations are consistent with the plume of groundwater representing the 
downstream output from reactions within the ore body. 

Micro-organisms were also observed in BH1. Aerobes or denitrifiers were not 
detected. Sulfate reducers and methanogens were recovered in enrichment cultures 
and the methane concentrations that were measured near 1 8 and 50 mbls suggested 
that H 2 produced within the ore body supports these microbial activities down- 
gradient. At depths between 50 and 60 m, the methane-bearing water appears to mix 
with sulfate-bearing water. Decreasing CH 4 and H 2 was accompanied by increasing 
S0 4 2 " and C0 2 . Although not stoichiometric, this relationship suggests that anaero- 
bic methane oxidation may occur in this zone. 

The alteration of the sulfide ore has induced the production of different gases: 
C0 2 , CH 4 , and H 2 , all of them participating in the biogeochemical cycles involved 
in the IPB decomposition. The observed characteristics of the underground miner- 
alogy, dominated by iron oxyhydroxides and sulfates, resulted from the alteration 
of the abundant sulfides of the IPB by chemolithotrophic micro-organisms (Figs. 
10.8 and 10.9). Sample analysis using scanning electron microscopy coupled with 
an energy-dispersive X-ray microanalysis probe (SEM-EDAX), in situ hybridiza- 
tion experiments, and direct cloning from deep core samples provide direct evidence 
of microbes metabolizing mineral substrates. As both secondary mineralogy and gas 
byproducts are the result of cryptic microbial communities living in the Rio Tinto 
acidic aquifer, they can be used as potential biomarkers to explore subsurface life 
in deep regions. 

In contrast to well-known AMD systems, the environments within and down- 
gradient from the Pena de Hierro VHMS appear to be anoxic, with a weakly acidic 
pH and evidence of methanogenic and sulfate-reducing activities. Any oxygen 
available from inflowing groundwater would initially be available as an electron 



10 Subsurface Geomicrobiology of the Iberian Pyritic Belt 



219 




I 1 nOum I 

Fig. 10.8 Scanning electron microscopy analysis of BH4 core sample 8,68c (162 mbls) 




I lOOpm I 

Fig. 10.9 SEM-EDAX iron mapping of Fig. 10.8 



220 R. Amils et al. 

acceptor for microaerophilic micro-organisms, as suggested by enrichment cultures 
results, but 2 could be also consumed by abiotic reactions (Chalk and Smith 1983; 
Conrad 1996). Because dissolved nitrate was not detected, quantities leached from 
the rock matrix are apparently consumed rapidly. Enrichment culture results sug- 
gest that some denitrifiers are present to utilize nitrate whenever it becomes 
available. 

Some of the spring waters down-gradient from the ore body are largely acidic, 
high in ferric iron, and red in color, as previously described (Fernandez-Remolar 
et al. 2003), which is typical of aerobic AMD processes. However, another group 
of springs found in the area (Fig. 10.3) produces anaerobic acidic waters with high 
concentration of ferrous iron. The origin of these iron-reduced springs remains to 
be determined. 



10.5 Conclusions 

The preliminary results from the MARTE project indicate that as groundwater 
enters the VHMS system, biotic and abiotic processes remove oxygen with the 
concomitant oxidation of iron and transient generation of acidity. Electron accep- 
tors available for microbial metabolism include oxygen, nitrite, nitrate, sulfate, 
ferric iron, and inorganic carbon. Electron donors include ferrous iron, sulfide, and 
hydrogen gas generated by water/rock interaction. This supports a population 
of microaerophilic and denitrifying autotrophs. As the fluids become more reduced, 
methanogenesis and sulfate reduction, using hydrogen gas, become the dominant 
microbial processes, and the pH rises. Oxidants to drive the system appear to be 
supplied by the rock matrix, in contrast to conventional AMD models. These 
resources need only groundwater to launch microbial metabolism. 

These observations confirmed the hypothesis that micro-organisms are active in 
the subsurface near the Rio Tinto headwaters. To our knowledge, this is the first 
observation of a subsurface ecosystem within an undisturbed VHMS rock environ- 
ment. The novel observations of H 2 and CH 4 production show that a variety of 
resources are available to support autotrophic microbial respiration in the subsur- 
face both within and down-gradient from the ore body. 

The Rio Tinto system may be an important model for astrobiological study 
(Fernandez-Remolar et al. 2003, 2005b; Amils et al. 2007). Recent observations of 
abundant layered sulfate minerals at Sinus Meridiani on Mars suggest a history of an 
aqueous, acidic, sulfate-rich environment (Squyres et al. 2004) that might originate 
from the weathering of sulfide-rich minerals (Fairen et al. 2004; Zolotov and Shock 
2005). Our results suggest that such a system, if the rocks were similar to those at 
Pena del Hierro, could support subsurface life, even if surface conditions preclude it. 
In addition, we found that subsurface microbial metabolism coupled to sulfide weath- 
ering can produce large amounts of methane, which has been suggested as an atmos- 
pheric indicator of extant life on Mars (Formisano et al. 2004). 



10 Subsurface Geomicrobiology of the Iberian Pyritic Belt 221 

Acknowledgements MARTE Project Team (participants are listed alphabetically): Angeles 
Aguilera, 1 Ricardo Amils, 1 ' 2 Carlos Briones, 1 Howard Cannon, 3 Fidel Davila, 1 Steven Dunagan, 3 
Alberto G. Fairen, 2 David Fernandez-Remolar, 1 Brian Glass, 3 Felipe Gomez, 1 Javier Gomez- 
Elvira, 1 Elena Gonzalez-Toril, 1 Lawrence G. Lemke, 3 Kennda Lynch, 4 Victor Parro, 1 Olga Prieto- 
Ballesteros, 1 Nuria Rodriguez, 1 Todd O. Stevens, 5 Virginia Souza-Egipsy, 1 Carol R. Stoker, 3 and 
Jhony Zavaleta. 3 ^entro de Astrobiologia (INTA-CSIC), Torrejon de Ardoz, Spain; 2 Centro de 
Biologia Molecular (UAM-CSIC), U. Autonoma de Madrid, Madrid, Spain; 3 NASA Ames 
Research Center, Mountain View, CA, USA, 4 NASA Johnson Space Center, Houston, TX, USA, 
Portland State University, Portland, OR, USA. 

We thank the following for their contribution to this work. Drilling services were provided 
by INSERSA S.A., Rio Tinto, Spain; fieldsites and laboratory space were provided by 
Fundacion Rio Tinto, Rio Tinto, Spain; logistic support was provided by Casiano Primo at the 
Hotel Vazquez Diaz, Nerva, Spain. We acknowledge the technical support provided by 
Mercedes Moreno-Paz, Marina Postigo, Maria Fernandez-Algar, and Moustafa Malki. 
Additional assistance was provided by Mary Sue Bell, James Hall, David McKay, Rachel 
Shelbe, and Norman Wainright. This work was supported by the NASA ASTEP program 
(USA), by institutional grants to the Centro de Astrobiologia (INTA-CSIC) and project 
CGL2006-02534/BOS from the Ministerio de Education y Ciencia. 



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Zavaleta J, Glass B, Lemke L (2004) Mars analog Rio Tinto Experiment (MARTE): 2003 
drilling campaign to search for a subsurface biosphere at Rio Tinto, Spain. Lunar and 
Planetary Science Conference, LPI contribution 1197, paper # 2025 

van Geen A, Adkins JF, Boyle EA, Nelson CH, Palenques A (1997) A 120-year record of wide- 
spread contamination from mining of the Iberian Pyritic belt. Geology 25:291-294 

Visviki I, Rachlin JW (1993) Acute and chronic exposure of Dunaliella salina and Chlamydomonas 
bullosa to copper and cadmium: Effects on growth. Arch Environ Contam Toxicol 26:149-153 

Visviki I, Santikul D (2000) The pH tolerance of Chlamydomonas applanata (Volvocales, Chlorophyta). 
Arch Environ Contam Toxicol 38:147-151. 

Wachtershauser G (1988) Pyrite formation, the first energy source for life: A hypothesis. System 
Appl Microbiol 10:207-210 

Widdel F, Schnell S, Heising S, Ehrenreich A, Assmus B, Schink B (1993) Ferrous iron oxidation 
by anoxygenic phototrophic bacteria. Nature 362:834-836 

Zolotov M, Shock E (2005). Formation of jarosite-bearing deposits through aqueous oxidation of pyrite 
at the Meridiani Planum, Mars Geophys Res Lett 32:L21203. doi: 10.1029/2005GL024253 



Chapter 11 

The Potential for Extant Life in the Soils 

of Mars 

Ronald L. Crawford(S) and David A. Newcombe 



11.1 Introduction 

In this chapter we discuss the present state of thought on the possibility that extant life 
in the form of micro-organisms may exist in the soils of Mars. The Viking missions of 
1976 have been the only experimental packages sent to Mars with the specific objective 
of searching for extant life in Martian soil samples. Landed missions since then have 
been geological packages that, although examining soils and rocks for minerals that 
might have biological origins, relied on instruments which were not designed to look 
specifically for living organisms. The Viking experiments provided some evidence for 
the possibility of life; however, the general scientific opinion (with notable exceptions; 
e.g., see http://mars.spherix.com/spie2/Spie2001Oxides/Spie2001-oxides.htm) has 
been that these experiments showed negative results (Klein 1992; Dick 2006). 

The existence in Martian soils of a hypothetical reservoir of problematic "highly 
oxidizing" material has been suggested in many scientific articles over the past 
quarter century. These putative oxidizing agents are said to be capable of destroying 
all organic matter and thus preventing the proliferation of life. However, experi- 
mental evidence from Mariner 9, Viking, Pathfinder (Rieder et al. 1997), Kitts 
Peak, Mars Global Surveyor (Hynek 2004), and the rovers Spirit (Haskin et al. 
2005; Gellert et al. 2004) and Opportunity (Rieder et al. 2004; Madden et al. 2004) 
indicate that Mars probably does not have an especially highly oxidative surface 
(http://mars.spherix.com/spie2/Spie2001Oxides/Spie2001-oxides.htm). The Martian 
regolith appears to harbor olivine-rich basaltic rock (Fig. 11.1) and materials such 
as the ferric sulfate mineral jaro site and gypsum. Some of these minerals appear to 
have been affected by water, indicating a mineralogy that is not too dissimilar 
from some basaltic systems that exist on Earth (Haskin et al. 2005). 

Microbes are ubiquitous in the dusts on Earth. The dusts covering most of the 
surface of Mars thus are potential carriers of microbes across vast areas of the 
Martian landscape (Hagen et al. 1970). Analyses of Martian dusts by instruments 



Ronald L. Crawford 

University of Idaho, Environmental Biotechnology Institute, P.O. Box 441052, Moscow, Idaho, 

83844-1052 

e-mail: crawford@uidaho.edu 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 225 

© Springer- Verlag Berlin Heidelberg 2008 



226 



R.L. Crawford and D.A. Newcombe 




Fig. 11.1 Photograph of a soil in Mars' Gusev Crater where the mineral olivine was detected by 
instruments aboard the Mars Exploration Rover Spirit. (Photo courtesy of NASA/JPL/Cornell; 
released January 20, 2004.) 



aboard the Mars Exploration Rovers indicate that there is nothing particularly 
remarkable about the chemistry of the dusts. They appear to be mostly of basaltic 
mineralogies, with some meteoritic material (Bandfield et al. 2003; Yen et al. 
2005). Carbonates, predominately MgC0 3 , have been spectroscopically identified 
at a level of 2-5% in Martian dust, and this mineral is expected to be thermo- 
dynamically stable on the surface of Mars (Quinn et al. 2006). 

Goetz et al. (2005) reported results from Mossbauer spectroscopy and X-ray 
fluorescence of dust particles captured from the Martian atmosphere by magnets 
carried by the Mars Exploration Rovers. The dust collected by the magnets was 
found to contain magnetite and olivine and some ferric oxides, indicating basaltic 
origins. Magnetite appears to be the mineral responsible for the magnetic properties 
of the dust. Thus, Martian dusts may be reasonably hospitable to extremophilic 
micro-organisms such as endospore formers of the genus Bacillus known on Earth. 
Unfortunately, as is generally the case for Mars, the unique properties of this planet's 
atmosphere complicate such generalizations. 



1 1 The Potential for Extant Life in the Soils of Mars 227 

According to Delory et al. (2006), laboratory studies, numerical simulations, and 
desert field tests indicate that aeolian dust transport can generate atmospheric electricity 
via contact electrification or "triboelectricity." Thus, electrically charged dust generated 
during dust storms on Mars could potentially provide ingredients for the generation of 
oxidants and thereby affect the habitability of Mars dusts and even the Martian surface. 
One oxidant that might be produced by this mechanism from traces of water in the 
atmosphere is hydrogen peroxide. Atreya et al. (2006) suggest that hydrogen peroxide, 
or a superoxide formed from reactions catalyzed by it, might scavenge organic material 
from the surface soils of Mars and thereby act as a biocide. However, this impact should 
be minimal on microbes shielded by burial in the subsurface. Thus, theories concerning 
the lethality of soils on Mars, especially questions regarding the potential effects of 
"Martian oxidants" on possible extant Martian life, remain mostly conjecture until more 
direct study of the Martian surface can be performed. 

Unfortunately, Mars is a very large place with many potential microbial niches. 
Research on Earths' harshest environments that might be considered as surrogates for 
Mars, such as the Atacama Desert of Chile, show that microbial life does exist, but 
microhabitats that harbor life are widely dispersed, are difficult to detect, and can be 
mostly hidden by lifeless surroundings (Warren-Rhodes et al. 2006; see Chapter 6). 
Viking clearly could simply have sampled such a lifeless area, missing a microniche 
harboring life a few centimeters, meters, or kilometers away. Thus, the Viking results 
cannot be considered definitive, even if it is accepted that they were negative. 

Based on research performed in the most extreme locations on Earth, potential 
sites for life in Martian soils include: buried water ice or permafrost (Titus et al. 
2003; Steven et al. 2006; Tung et al. 2005; see Chapters 7 and 12), the deep sub- 
surface (Weiss et al. 2000; see Chapter 10), and within protected layers of rocks 
(Kuhlman et al. 2005; see Chapter 6). We do not examine the existing controversies 
about evidence for past life on Mars, such as the on-going arguments over the origin 
of magnetite in Martian meteorite ALH84001 (Weiss et al. 2004). Instead, we 
examine the question, "Is it reasonable to expect to find extant life in the soils of 
Mars were we to have the resources and technologies needed to look wherever we 
desired on or below the Martian surface?" 



11.2 Mars as a Microbial Habitat 

11.2.1 Mars Is an Exceedingly Harsh Environment 

At first glance, by Earthly standards Mars does not constitute a favorable environ- 
ment for terrestrial micro-organisms. The environmental stresses on Mars include 
low temperature (-123°C to 25°C), low-pressure (-600 Pa = 6mbar), high fluxes of 
biologically destructive, low-wavelength ultraviolet (UV) irradiation (8.4 to 67 W/m 2 ; 
Diaz and Schulze-Makuch 2006), and perhaps a highly oxidizing environment 
(Crawford et al. 2003; Krai et al. 2004; Benner et al. 2000). Also, inasmuch as Mars 
has no magnetic field and a very thin atmosphere, galactic cosmic rays and solar 



228 R.L. Crawford and D.A. Newcombe 

flare particle fluxes may impact and penetrate the surface to a greater extent than 
they do on Earth. Potentially lethal doses of these types of radiation may accrue on 
the Martian surface and even to some depth in the soil. However, such ionizing 
radiation does not appear to be sufficient for sterilizing soil at least in the short term 
(for a discussion of this topic, see http://www7.nationalacademies.org/ssb/ 
bcmarsch3 .html) . 

Recently, researchers analyzing data from the Electron Reflectometer (ER) on 
board the Mars Global Surveyor noted areas in which a dropoff in electron flux 
measured by the ER was observed (Mitchell et al. 2001). A surficial map of the 
readings corresponded to localized intense crustal magnetic fields. These fields are 
strong enough to stave off solar winds allowing their measurement with ER on a 
spacecraft 400 km above the surface and raising the question as to whether the 
fields may be strong enough to provide zones of reduced energetic particles and 
thereby moderate deleterious ionizing properties. If so, could these local zones be 
more favorable to microbial survival? 

Even highly stress-resistant microbes such as Deinococcus radiodurans and the 
cyanobacterium Chroococcidiopsis do not survive very long in near-surface soils 
under the combined environmental stresses of low temperature, ultra-low-pressure, 
and intense ultraviolet irradiation (Diaz Schulze-Makuch 2006; Cockell et al. 
2005). However, survival of such organisms can be greatly enhanced by the 
presence of liquid water and burial at depth in the soil. Thus, the subsurface of Mars 
is likely to be to be a far more favorable environment than the surface for survival 
and proliferation of micro-organisms such as those we know from Earth. 



11.2.2 The Atmosphere of Mars Is Thin and Unlike 
That on Earth 



The atmospheric composition of Mars is not Earth-like. The atmosphere of Mars 
contains mostly carbon dioxide (C0 2 , -95%) with some nitrogen (N 2 , -2.7%), 
argon (Ar, -1.6%), oxygen (0 2 , -0.13%), and traces of water vapor (H 2 0, -0.03%; 
Soffen 1976; Clancy et al. 1990). There are small amounts of methane (CH 4 ) in the 
Martian atmosphere. The global average methane mixing ratio, as measured by 
the Planetary Fourier Spectrometer on the European Mars Express spacecraft, was 
10 ± 5 parts per billion by volume (ppbv), varying between and 30ppbv over the 
planet (Formisano et al. 2004). There are other trace gases in the Martian 
atmosphere, including hydrogen (H 2 ) and carbon monoxide (CO) (Weiss et al. 
2000; Clancy et al. 1990). The composition of the atmosphere on Mars by itself 
may not be detrimental to the survival and proliferation of some Earth-like 
microbes. For example, the cyanobacteria Synechococcus and Anabaena were 
shown to survive 101 kPa (100%) pC0 2 when pressure was gradually increased by 
15kPa per day, and Plectonema actively grew under these conditions. All of these 
strains grew in an anoxic atmosphere of 5kPa pC0 2 in N 2 (Thomas et al. 2005). 
The composition of the gases within soils at depth on Mars is not known and may 
be locally different than those measured on the surface by the Viking spacecraft. 



1 1 The Potential for Extant Life in the Soils of Mars 229 

11.2.3 All Life Must Have a Source of Energy 

Any form of extraterrestrial life must have a source of energy (Crawford et al. 2002, 
2001; Lang et al. 2001), but this does not appear to be a problem on Mars (Irwin 
and Schulze-Makuch 2001; Chyba and Phillips 2002; Jakosky and Shock 1998). As 
Weiss et al. (2000) state, "The location and density of biologically useful energy 
sources on Mars will limit the biomass, spatial distribution, and organism size of 
any biota." On the surface there is of course adequate energy available from 
sunlight to support photo synthetic life forms that could use readily available carbon 
dioxide as a source of carbon. It is unlikely that organisms could use this radiant 
energy directly because of the extreme conditions on the surface (Weiss et al. 
2000), unless they were able to find protected niches within the soil subsurface 
(Crawford et al. 2003) or in other locations such as internal layers of rocks 
(Kuhlman et al. 2005). Other potential sources of energy for Martian biota include 
hydrothermal and chemical weathering energy (Jakosky and Shock 1998; Varnes et al. 
2003), thermo synthesis in the presence of a thermal gradient within ice (Muller 
2003), and photochemically produced atmospheric H 2 and CO diffusing into the 
regolith to protected regions of the subsurface. Based on modeling experiments, 
the latter energy source appears sufficient to sustain a subsurface CO/H 2 oxidizing 
microbial community on Mars (Weiss et al. 2000). 

11.2.4 Life as We Know It on Earth Requires Sources 
of Nitrogen and Sulfur 

For life forms as exist on Earth, nitrogen is required as a nutrient for the synthesis 
of proteins, nucleic acids, and various metabolic cofactors. Sulfur is required for 
synthesis of certain amino acids and enzyme cofactors. Thus, if microbes similar to 
those found on Earth exist on Mars, they must be able to access sources of these 
two elements. As discussed above, some nitrogen (N 2 , -2.7%) is found in the 
atmosphere of Mars (Soffen 1976; Clancy et al. 1990). Thus, extant microbes in 
Martian soils might access this nitrogen supply by complex processes similar to 
those used by free-living nitrogen-fixing bacteria on Earth (Peters et al. 1995). 
Measurement of nitrogen on present-day Mars has been limited to only that present 
in the atmosphere, and this atmospheric nitrogen represents a small fraction of the 
nitrogen thought to have been received by the planet during its formation 
(Mancinelli and Banin 2003). As hypothesized by Mancinelli and Banin (2003), 
Mars soils may be like soils seen in Earth's extremely dry deserts. Such soils 
contain some nitrogen as nitrate salts and some as fixed ammonium bound to 
alumino silicate minerals. Analyses carried out on the Martian surface by the 
Opportunity Rover show that rocky outcrops are rich in sulfur (Reider et al. 2004; 
Haskin et al. 2005). Thus, sufficient nitrogen and sulfur probably are available to 
support extant microbial life in the soils of Mars. Capone et al. (2006) suggest that 
analysis of the abundance and chemistry of nitrogen deposits will provide important 
clues as to the presence of life. 



230 R.L. Crawford and D.A. Newcombe 

11.2.5 Could There Be Methanogens on Mars? 

Krai et al. (2004) and others (Boston et al. 1992; McCollom 1999) suggest that 
Martian equivalents of Earth's methane-forming bacteria (methanogens) might be 
a type of micro-organism that could take advantage of the particular energy supplies 
available in the Martian subsurface or on other extraterrestrial locations such as 
Europa. As Krai et al. (2004) discuss, methanogens might be able to use a geother- 
mal source of hydrogen for energy, which might be provided by volcanic or 
hydrothermal activity or the reaction of basalt and anaerobic water. For carbon they 
could use C0 2 , which is abundant in the Martian atmosphere and soil. In addition, 
they would need subsurface liquid water. Krai et al. (2004) performed experiments 
to show that certain methanogens can grow on a Mars soil simulant when supplied 
with C0 2 , H 2 , and varying amounts of water. Thus, methanogens can grow even in 
relatively nutrient-poor soils, as long as liquid water is available, even if water 
availability is intermittent (Kendrick and Krai 2006). 

Evidence is slowly accumulating from satellite-based observations such as those 
of the European Space Agency's Mars Express, which carries the Advanced Radar 
for Subsurface and Ionospheric Sounding (MARSIS) instrument, that ice and 
perhaps subsurface water do exist on Mars. MARSIS detected radar reflections 
from a subsurface base of an ice layer close to the planet's north pole, indicating 
that the deposit is about 1.8-km thick (Schilling 2005). Another radar instrument 
(SHARAD, Shallow Subsurface Radar) was launched on NASA's Mars 
Reconnaissance Orbiter that has been in orbit since March 10, 2006 and will greatly 
enhance the search for subsurface liquid water (Reichhardt 2005). SHARAD will 
seek liquid or frozen water at up to 1 km into Mars' crust (see also Section 11.3.1 
for a discussion of recent evidence for liquid water presence). 

Rother and Metcalf (2004) remind us that certain methanogens can use carbon 
monoxide as a growth substrate, producing methane via a pathway that involves 
hydrogen as an intermediate. They tested the ability of Methanosarcina acetivorans 
C2A to use CO as a growth substrate, finding it to grow on CO to high cell densities 
with a doubling time of only 24 h. Methane formation surpassed acetate and formate 
formation when the cultures entered a stationary growth phase. As discussed above, 
CO is available to some degree in the Martian environment (Weiss et al. 2000), so 
the CO/H 2 combination for support of methanogenic growth appears feasible on Mars. 
This possibility is further suggested by the observation that hydrogen-based metha- 
nogenic communities do occur in Earth's subsurface, providing an analogue for 
possible subsurface microbial ecosystems on other planets (Chapelle et al. 2002). 

How likely is an extant "methanogens on Mars" scenario? As mentioned above 
(see Section 11.2.2), Formisano et al. (2004) recently reported the detection of 
methane in the Martian atmosphere at a global average methane mixing ratio of 10 
± 5 ppbv. They concluded that the source of methane could be either biogenic or 
nonbiogenic, including past or present subsurface micro-organisms, hydrothermal 
activity, or as a result of cometary impacts. Others have confirmed the presence of 
methane in the Martian atmosphere (Krasnopolsky et al. 2004; Mumma et al. 2004). 



1 1 The Potential for Extant Life in the Soils of Mars 23 1 

Onstott et al. (2006) mathematically modeled the Martian subsurface methane flux 
based on available data from Mars using previous information about methane 
sources within the South African Precambrian crust of the Witwatersrand Basin. 
They concluded that methane should only reach the surface if it is found as a 
hydrate and the hydrate is saturated in the cryosphere; otherwise they suggest it 
should be captured within the cryosphere. The sublimation of such a hydrate-rich 
cryosphere could generate the observed methane flux. 

They also conclude that a microbial explanation of methane production only 
seems possible if there is a hypersaline environment above the hydrate stability 
zone. Methane as a hydrate can be derived from a number of sources, so Onstott 
et al. (2006) suggest that the C and H isotopic values of Martian CH 4 be analyzed 
by a future Mars instrument package, which would be one of the better ways to look 
for the possibility of an Earth-like biotic signature. Thus, there appears to be 
measurable methane in the Martian atmosphere, and it seems to be replenished 
continuously, perhaps from the subsurface. Indeed, methane survives for a relatively 
short time (a few hundred years) in the Martian atmosphere so it must be constantly 
replenished in order to maintain the observed concentrations. Its origin (biotic or 
abiotic) will remain an unanswered question until more analyses can be performed 
using instruments landed on the Martian surface or passed through its atmosphere 
(see also Webster 2005; Durry et al. 2004; de Bergh 1995). 



11.3 Searching for Microbes in the Soils of Mars 

11.3.1 Where Should Investigators Look for Extant 
Life on Mars? 

In order to increase the likelihood of success in finding extant micro-organisms on 
Mars, it will be crucial to carefully consider where to look. This led Klein (1992) 
to state that, "Attempts to search for extant biology should be restrained until 
adequate new information about potential habitable microenvironments is obtained." 
In the last decade, considerable progress has been made toward this goal. Several 
locations where life might exist on Mars have been suggested (Rothschild 1990). 
The most commonly mentioned niches for life on Mars include those listed below. 
The common feature of all these environments is that they are to some degree 
protected from surrounding harsh conditions and/or support the availability of 
liquid water (Farmer et al. 1995). 

• In or on rocks: This is a niche analogous to that of Earth's endolithic cyano- 
bacteria that live inside porous sandstone rocks, protected by a thin rock crust 
(Friedmann and Ocampo-Friedmann 1984) or evaporates (Rothschild 1990). 

• Caves: The field of cave geomicrobiology has direct relevance to studies of 
extant life on other planets. Caves may provide protection from surface 



232 R.L. Crawford and D.A. Newcombe 

stresses such as UV radiation. Caves on Earth contain many unusual organisms 
that oxidize or reduce minerals such as manganese, iron, and sulfur (Boston 
et al. 2003). 

• In thin mineral "varnishes": This niche is represented by the thin layer of silica 
and metal oxides that frequently covers rocks in virtually all of Earth's dry and 
cold deserts (Kuhlman et al. 2005). 

• In polar ice caps: This niche would be similar to Earthly locations where snow 
and ice algae are found (Kohshima 2000). 

• In buried water ice or permafrost (Steven et al. 2006; Tung et al. 2005). 

• In possible volcanic regions: This environment would be similar to regions near 
deep-sea hydro thermal vents where some of Earth's chemolithoautotrophs are 
found (Miroshnichenko and Bonch-Osmolovskaya 2006; Gaill 1993). 

• In the generic "deep subsurface" (Weiss et al. 2000). 

• In areas of nitrogen salt accumulation (Mancinelli and Banin 2003; Capone 
et al. 2006). 

• In areas maintaining an intense localized magnetic field (Mitchell et al. 2001). 

Images taken by the Mars Global Surveyor Mars Orbiter Camera (MOC) 
suggest that liquid water existed on Mars in the recent past (Fig. 11. 2A). 
Observations of the same Martian surface location in 1999 and then in 2005 further 
indicate that liquid water may have flowed on the planet's surface during the last 
seven years (Fig. 1 1.2B). Such images are very useful in planning landing locations 
for future life detection missions on the planet. 

Ostroumov (1995) suggests that any viable micro-organism on Mars probably 
exists with minimum metabolism in compact zones and that these zones may contain 
microvolumes of unfrozen water in the Martian permafrost. Such zones, if they 
exist, are likely to be located deep beneath the Martian surface (Weiss et al. 2000) 
and may be widely dispersed and difficult to locate (Warren-Rhodes et al. 2006). 

Other less obvious locations might also be considered. For example, 
Rothschild (1990) points out that micro-organisms can survive in salt crystals 
and actively metabolize while encrusted in evaporites. Evaporites may occur on 
Mars and can attenuate UV radiation while transmitting light that might be used 
for photosynthesis (light of 400-700 nm in wavelength). Thus, this author 
proposes that evaporites might provide a niche for extant Martian microbial 
communities on Mars. Ellery and Wynn-Williams (2003) also suggest that 
evaporites in paleolake craters might be a good place to look for life on Mars. 



11.3.2 How Can Investigators Obtain Appropriate Samples 
of Martian Soil to Look for Extant Life? 

As mentioned in the preceding section and based on what is now known about 
potential niches for microbial life in the soils of Mars, it appears that the best places 
to look will be located well below the surface of the planet (Reichhardt 2005) in 



1 1 The Potential for Extant Life in the Soils of Mars 



233 





Fig. 11.2 (A) Gully landforms proposed to have been caused by geologically recent seepage and 
runoff of liquid water on Mars' south polar pitted plains (photo from the Mars Global Surveyor 
Mars Orbiter Camera (MOC) courtesy of NASA/JPL/Malin Space Science Systems). This image 
was acquired July 14, 1999 and covers an area approximately 2.8 km wide by 2.1km high. (B) 
NASA photographs taken by the MOC have revealed bright new deposits observable in two gullies 
that suggest water carried sediment through them sometime during the past seven years (image 
released December 6, 2006) 



234 R.L. Crawford and D.A. Newcombe 

niches that are protected from stresses such as UV light (e.g., caves) or in areas 
where strong magnetic fields are present that attenuate galactic cosmic rays and 
solar flare particle fluxes (http://www7.nationalacademies.org/ssb/bcmarsch3. 
html). It also will be crucial to explore sites where liquid water is likely to reside 
at least intermittently. 

Assuming that interdisciplinary teams of scientists and engineers can choose 
several logical locations to search (Lobitz et al. 2001), the next problem becomes how 
to aseptically sample at these places. Because the best sampling locations may be 
multiple kilometers below the surface, the challenges will be daunting. Blacic 
et al (2000) analyzed some of the challenges associated with aseptic drilling and 
sampling on Mars to a depth of 200 m, which may not be deep enough to explore 
some of the most likely habitats for microbial life. Their analyses eliminated all 
known terrestrial drilling technologies but provided some ideas for approaches that 
might have promise. The NASA Ames Research Center, Honeybee Robotics, Georgia 
Tech, and the Mars Institute are involved in the Drilling Automation for Mars 
Exploration (DAME) project, focused on development of automated technologies for 
future drilling exploration of Mars. The team has developed a Mars-prototype drill 
that is lightweight, uses no lubricants, and needs relatively little power. Honeybee 
Robotics is building the drill that is being tested in the regolith-like material inside 
Haughton Crater on Devon Island in Canada's Nunavut Territory north of Ontario and 
Quebec. This activity involves drilling into ice layers and permafrost similar to what 
one might expect to find near the surface in Martian polar regions (http://www.nasa. 
gov/centers/ames/research/exploringtheuniverse/ai .html) . 

NASA is already field-testing another system designed to drill for subsurface 
Martian life. The drill called MARTE includes a drilling platform and suite of 
scientific instruments that are able to search for evidence of life in samples the 
robotic drill has extracted from below ground. The drill rig is about 2.4-m tall and 
sits on a three-legged platform about 2.1m in diameter. The drill uses less than 
150W of power, bores using carbide-diamond cutters, and uses no drilling fluid. 
It makes core plugs of rock that are about 20-cm long and brings them to the surface 
(http://marte.arc.nasa.gov/). The drill only reaches depths of about 6 m, so if used 
on Mars, it would have to be employed at locations where extant life might be 
relatively near the surface (e.g., within caves or fields of evaporites). 

One promising approach for sampling beneath the Martian surface would be 
through the use of a torpedolike device, or "CryoScout," that could melt its way 
through the ice cap on Mars (Kounaves 2003) and deliver samples to an instrument 
carried along or to the surface for analysis. Ellery et al. (2005) propose "Vanguard," 
a spacecraft that might be transported to Mars by a Mars Express-type spacecraft 
and would carry ground-penetrating, instrument-equipped "moles" mounted onto a 
rover for subsurface penetration but only to a depth of about 5 m. Each mole would 
take a one-way trip down a borehole and provide real-time data without the need 
for recovery of moles or samples. 

Mancinelli (2003) suggests that a manned mission may be necessary for 
sampling the deep Martian subsurface. Such a mission could involve drilling 3 km 
or more into the Martian soil, collecting samples, and conducting preliminary 



1 1 The Potential for Extant Life in the Soils of Mars 235 

analyses to select samples for return to Earth. This scenario, as others presented 
above, requires that the drilling equipment be sterilized prior to use; the collection, 
containment, and retrieval of samples would need to be conducted such that the 
mission crew is protected from exposure to the collected soils or their dusts. Also, 
Martian soils (or ice) returned to Earth must be physically and biologically isolated 
from the time they are collected until analysis inside a BL4-level containment 
facility on Earth. The presence of humans on Mars would clearly make meeting 
these difficult challenges more feasible. A manned mission of this magnitude is 
probably decades away, but scientists are already thinking about how to circumvent 
the many environmental and engineering challenges involved in such a mission 
(Ehlmann et al. 2005). Even on Earth, sampling microbial communities in the deep 
subsurface, without introducing contaminants from the surface or from human 
sources, is very difficult (see Chapter 10). Extreme measures and complex controls 
must be used to prove that sampling has indeed been accomplished without con- 
tamination (Lehman et al. 2001; Juck et al. 2005). Clearly, trying to sample from 
the deep subsurface of Mars while avoiding contamination by Earthly microbes 
brought along by astronauts or aboard robotic spacecraft will be an even greater 
challenge. 



11.4 Detection of Life in Martian Soils 

11.4.1 Analytical Methods Are Highly Developed for Detecting 
Organic Signatures of Life and Life Processes in Soil 

Once investigators have decided where to look for life in Martian soils, then methods 
must be employed to detect any extant life present. Suggested approaches are many 
and varied and date from the 1960s (Levin et al. 1964; Christian et al. 1965) prior 
to the Viking missions to the present time. For example, Simmonds (1970) 
discussed pyroly sis-gas chromatography-mass spectrometry as a life detection 
method, and the science payload on Viking included a combined pyrolysis-gas 
chromatography-mass spectrometry instrument to detect thermal fragments 
originating from the principal classes of bio-organic matter found in living systems 
such as protein and carbohydrate. No such molecules were observed. This could 
mean that life does not exist on Mars, that the wrong location was tested, or that 
the detection limit of the instrument was insufficient and organic molecules were 
actually present but were missed or were invisible to the GC-MS used (Benner 
et al. 2000). Nonetheless, it is likely that chromatography-mass spectrometry will 
be a mainstay of extraterrestrial life detection experiments in the future because 
instruments have been greatly improved and miniaturized in the post- Viking era. 
Investigators also now know much more about Mars and what to look for as 
compared to the state of knowledge three decades ago (Palmer and Limero 2001; 
Pietrogrande et al. 2005). 



236 R.L. Crawford and D.A. Newcombe 

A related method for chromatographic separation and detection of organic 
molecules extracted from soil is capillary electrophoresis, which has been developed 
as a useful miniaturized tool for examination of Martian soils. This technique involves 
chiral separations of fluorescein isothiocyanate-labeled amino acids using a 
microfabricated capillary electrophoresis chip, and it has been proposed to explore the 
feasibility of using such devices to search for extinct or extant life signs in 
extraterrestrial environments (Hutt et al. 1999). Also, Lang et al. (2001) used 
supercritical fluid extraction and both capillary electrophoresis and high-performance 
liquid chromatography (HPLC) equipped with diode array or electrochemical 
detectors as a means to detect signature biological redox compounds as an approach 
for the detection of molecules that could be signatures of life in extraterrestrial soils. 



11.4.2 Radiorespirometry Is a Highly Useful and Sensitive 
Life Detection Technique 

Levin et al. (1964) developed a radiorespirometric approach for life detection. This is 
a highly logical approach for detection of actively metabolizing heterotrophic 
microbes and involves the introduction of a soil sample into a microbiological 
medium that supports growth of a wide range of Earth micro-organisms. In this case, 
however, selected ingredients of the medium are labeled with radioactive 14 C. If these 
compounds are degraded (mineralized) to carbon dioxide, the C0 2 evolved will 
be tagged as 14 C0 2 , which can be detected easily with great sensitivity. In one of the 
classic experiments of modern times, such a radiorespirometric instrument was 
landed on Mars aboard Viking; 14 C0 2 was in fact produced from a sample of Martian 
soil. However, this result is now thought by most in the scientific community to have 
resulted from abiotic oxidation of the substrates by strong "Mars soil oxidants" (Klein 
1992), although the discussion continues (Van Dongen et al. 2005). 



11.4.3 Life Might Be Detected by Observing Controlled Electron 
Transport Used by Living Organisms to Obtain Useful Energy 

Lang et al. (2001) suggested a method for life detection based on the fact that living 
entities require a continual input of energy accessed through coupled oxidations and 
reductions (an electron transport chain). They demonstrated using Earthly soils that 
the identification of extracted components of electron transport chains is useful for 
remote detection of a chemical signature of life. The prototype instrument package 
these investigators developed used supercritical carbon dioxide for soil extraction, 
followed by chromatography or electrophoresis to separate extracted compounds, 
with final detection by voltammetry and tandem mass spectrometry. Later, the same 
group used Earth-derived soils to develop a related life detection system based on 
direct observation of a biological redox signature (Crawford et al. 2002). 



1 1 The Potential for Extant Life in the Soils of Mars 237 

They measured the ability of soil microbial communities to reduce artificial 
electron acceptors. Living organisms in pure culture and those naturally found in soil 
were shown to reduce 2,3-dichlorophenol indophenol (DCIP) and the tetrazolium 
dye 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner 
salt (XTT). Uninoculated or sterilized controls did not reduce the dyes. A soil from 
Antarctica that was determined by chemical signature and DNA analysis to be sterile 
also did not reduce the dyes. Dye reduction was readily monitored by observing 
dye color changes upon reduction, a simple approach to implement robotically. 
The authors concluded that observation of dye reduction, supplemented with 
extraction and identification of only a few specific signature redox-active biochemi- 
cals such as porphyrins or quinones could provide a simplified means to detect a 
signature of life in the soils of other planets or their moons. 

Direct microscopic visualization of bacteria in collected subsurface rock speci- 
mens might be another alternative for detection of Earth-like microbes. Tobin et al. 
(1999) developed a protocol that enables the visualization of intact microbial cells 
in petrographic thin sections that might be adaptable to a Mars mission. Their 
method avoided detaching the cells from their host mineral surfaces and avoided 
microbial contamination during the lapidary process. Nucleic acid stains that 
specifically target double- stranded DNA and RNA were utilized for in situ visuali- 
zation of cells in surface and subsurface basalts collected in Idaho, USA. Extending 
this approach, the authors found that examination of samples incubated with acetic 
acid-UL- 14 C using phosphor imaging allowed in situ visualization of 14 C-labeled 
biomass. The greatest challenge for this type of direct visualization technology will 
be its detection limit. Microbial distribution in Martian rocks likely would exhibit 
a high degree of spatial heterogeneity at the micrometer scale, and this could easily 
lead to failure in observing microbes that are actually present. As with many 
techniques discussed in this chapter, a positive result would be outstanding but a 
negative result still ambiguous. 

Not every paper on this topic can be discussed here, but representative publica- 
tions describing a variety of approaches proposed for detection of signatures of life 
in Martian soil are summarized in Table 11.1. 



11.5 Conclusions 

By Earthly standards, Mars is clearly not a favorable environment for terrestrial 
micro-organisms and so differs from environments across much of our own planet. 
The most biologically significant stresses on Mars include low temperature, high 
fluxes of biologically destructive radiation, possible shortages of certain nutrients 
such as nitrogen, and perhaps a highly oxidizing and therefore biocidal surface. 
In contrast, the geochemistry of the rocks and soils on Mars based on observations 
by orbiting and landed spacecraft over the past 35 years indicates a mineralogy not 
too dissimilar from some basaltic systems seen on Earth that are well colonized by 
micro-organisms. Also, Mars has sufficient sources of carbon and energy to support 



238 



R.L. Crawford and D.A. Newcombe 



Table 11.1 Methods suggested for the detection of extraterrestrial life 



Proposed Life Detection Methods 



References 



Pyrolysis-gas chromatography-mass 

spectrometry 
Gas chromatography-mass spectrometry 

Detection of controlled electron transport 
and signature biological redox compounds 

Fluorescence techniques 

Phosphatase activity 

Immunological approaches 

Raman spectroscopy 

Remote sensing of chlorophylls 

Dichroism spectroscopy 

Electrochemical and 
polarimetric methods 

Observations of structural complexity 

Detection of amino acids 

Use of charge-coupled devices 

Release of heat from metabolizable substrates 

as measured by a microcalorimeter 
Determination of optical activity (turbidity) 

from growth on organic substrates 
Soil gas disequilibria 

In situ imaging 



Simmonds (1970) 

Fox (2002); Buch et al. (2003); 

Pietrogrande et al. (2005) 
Sotnikov (1970); Crawford et al. (2002); 

Lang et al. (2001) 
Sotnikov (1970); Kawasaki (1994) 
Kobayashi et al. (2004) 
Schweitzer et al. (2005) 
Ellery and Wynn- Williams (2003) 
Knacke (2003) 
Xu et al. (2003) 
Thiemann (1975); Kounaves (2003) 

Nealson et al. (2002) 

Pollock et al. (1977); Hutt et al. (1999); 

Rodier et al. (2001) 
Nussinov et al. (1992) 
Imshenetsky et al. (1976) 

Imshenetskii and Evdokimova (1975) 

Brazhnikov et al. (1971); 

Kelley et al. (1975) 
Tobin et al. (1999) 



the growth of a large variety of Earth-like micro-organisms. Based on decades of 
research performed in the most extreme locations on Earth, the potential for extant 
microbes in the soils and rocks of Mars cannot yet be excluded. Potential sites for 
life on Mars include: in buried water ice or permafrost, within the deep subsurface, 
within protected layers of rocks or in caves, in possible volcanic regions, in areas 
of nitrogen salt accumulation, and in areas maintaining an intense localized 
magnetic field that may protect life from some forms of ionizing radiation. 

Based on these generalizations, it is reasonable to expect to find extant life in 
Martian soils if investigators were able to apply the resources and technologies 
needed to look wherever desired, particularly below the Martian surface. The oppo- 
site hypothesis, that Mars is sterile, likewise cannot be dismissed. Unfortunately, 
scientists at this point have too little information to make a definitive conclusion as 
to the existence of extant life on Mars. This relegates the scientific community to 
continued discussion and speculation until humans make the trip and look first- 
hand or until appropriately collected and processed samples of Martian soils and 
rocks are returned to Earth for careful examination. Thus, the question posed by the 
title of this chapter remains open for debate. 



1 1 The Potential for Extant Life in the Soils of Mars 239 

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Chapter 12 

Bacteriology of Extremely Cold Soils Exposed 

to Hydrocarbon Pollution 

Lucas A.M. Ruberto, Susana C. Vazquez, and Walter P. Mac Cormack(K) 



12.1 Introduction 

The study of bacterial communities present in natural ecosystems has been the 
object of attention of several research groups during the past 15 years, driven 
mainly by the development of DNA-based methodologies (Amman et al. 1995; 
Holben 1997). Taking advantage of these methods, it was shown that most of the 
individual components of natural bacterial communities are incapable of growth on 
standard culture media. In this sense, it was evident that more than 99% of the bac- 
terial cells present in a soil sample were not able to grow on standard culture media 
(Roszak and Colwell 1987; Torsvik et al. 1990). Soil represents an extremely com- 
plex matrix. Soils considered as habitat are characterized by physical, chemical, 
and temporal heterogeneities at any scale from km to nm (Young and Ritz 2000). 
Each field, forest, tundra, or cold desert has a unique soil food web with a particular 
proportion of organisms, and a particular level of complexity within each group of 
organisms. These differences are the result of soil, vegetation, and climate factors. 

Detailed studies on soil micro-organisms demonstrated that even within soil 
habitats deemed homogeneous at the plot scale, distribution of soil bacteria was 
highly structured, with different bacterial communities located in specific sites, 
probably in response to the heterogeneities imposed by the habitat (Franklin and 
Mills 2003). Although the above-cited references dealt with agricultural soils, the 
concept of soil heterogeneity is a general rule (Young and Crawford 2004) and must 
be kept in mind in all descriptive or analytical studies of soil bacterial communities. 
Bacterial populations located in individual soil microhabitats perform several 
biophysical and biochemical processes, turning the soil into the most complex and 
biodiverse ecosystem on Earth. 

As was mentioned above, local climate conditions are among the main factors 
conditioning the number and type of micro-organisms present in a soil. In Antarctica 
(as well as in the Arctic, the Alps, the Puna, and other high-altitude soils), low 



Walter P. Mac Cormack 

Institute- Antartico Argentino, Departamento de Biologia. Cerrito 1248 (C1010AAZ), Buenos 

Aires, Argentina 

e-mail: wmac@huemul.ffyb.uba.ar 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 247 

© Springer- Verlag Berlin Heidelberg 2008 



248 L.A.M. Ruberto et al. 

temperature represents a key selection-pressure factor restricting microbial diversity. 
In these extremely cold soils, only psychrotolerant and psychrophilic micro-organisms 
are able to survive and proliferate (Nedwell 1999; Deming 2002). 

The presence of contaminant chemicals from anthropogenic origin represents an 
additional factor of stress that strongly affects the composition of the microbiota of 
contaminated soil. These changes were reported for soils and sediments from dif- 
ferent areas of the world (Roling et al. 2002; Haack et al. 2004; Zocca et al. 2004; 
Katsivela et al. 2005) including Alpine (Margesin et al. 2003), Arctic (Juck et al. 
2000), and Antarctic (Mac Cormack et al. 1998; Aislabie et al. 2001; Saul et al. 2005) 
extremely cold soils altered by hydrocarbon contamination. Although Antarctica is 
at the present time one of the sites least affected by human activity worldwide, the 
scientific and logistic stations (which are powered almost exclusively by combus- 
tion of oil derivative fuels) as well as the fishery and tourist activities (the latter 
having increased dramatically over the last years) have led to spatially limited 
hydrocarbon contamination that in some cases has been highly significant 
(Kennicutt II and Sweet 1992; Aislabie et al. 2004). Most of the Antarctic sites 
where stations are built and which are consequently at high risk of hydrocarbon 
pollution are restricted to those small areas remaining free of ice during the summer. 
Together, these areas represent less than 0.3% of the Antarctic surface, which is 
almost 14,000,000 km 2 in total (Fox and Cooper 1994). They occur mainly in the 
shore of the Antarctic Peninsula and the Ross Sea region. Humans are not the only 
living beings that chose these areas to settle down. On the contrary, the major part 
or the Antarctic micro- and macroorganisms develop in these ice-free coastal areas, 
including the only two vascular plants found in Antarctica and the penguins, sea- 
birds, and several marine mammals that arrive in spring for mating and breeding. As 
a consequence of this, pollution in Antarctica does not affect humans only, but it is 
a global environmental problem that probably has a deep effect on soil food webs, 
which are much simpler and more fragile than those encountered in other biomes 
of the world. In addition to a direct effect on plants and animals, hydrocarbons may 
also modify the entire terrestrial food web by altering the soil microbiota, changing 
the type of species being present and the number of their representatives. 

In the last decades, bioremediation - or the exploitation of the ability of micro-organ- 
isms (mainly bacteria) to catabolize hydrocarbons and other organic pollutants - has 
been considered as the most adequate tool for reducing contaminant levels and 
eventually clean up affected areas. Studies on variously located soils have been 
published, documenting the effectiveness of different bioremediation strategies for 
reducing soil hydrocarbon contamination (Heitzer and Sayler 1993; Hooker and 
Skeen 1996; Cunningham and Philp 2000; Marchal et al. 2003; Bento et al. 2005). 
These studies have mainly dealt with the extremely cold soils from the Arctic (Whyte 
et al. 1999; Eriksson et al. 2001; Mohn et al. 2001; Rike et al. 2003) and the Alps 
(Margesin and Schinner 1997a,b, 2001). Recently, soils from Antarctica have received 
some attention (Delille and Pelletier 2002; Ruberto et al. 2003; Delille et al. 2004; 
Snape et al. 2005). In all these cold soils, the presence of efficient hydrocarbon- 
degrading bacterial isolates was reported, such organisms constituting communities 
selected from the original soil inhabitants after a long history of contamination 
(Mac Cormack and Fraile 1997; Whyte et al. 1997; Thomassin-Lacroix et al. 2001; 



12 Bacteriology of Hydrocarbon-Contaminated Cold Soils 249 

Baraniecki et al. 2002; Margesin et al. 2003). However, the low level of metabolic 
activity prevailing at low temperatures, the lower evaporation rate of the volatile 
compounds compared with rates observed for temperate soils, and even the existence 
of a permafrost and an active layer under continuous cycles of freezing and thawing 
influence the fate of the different hydrocarbons and must be added to the list of 
known factors already identified as interfering with degradation of contaminating 
hydrocarbons in all soil types (Providenti et al. 1993). 

Due to the limitations imposed on bacterial growth by the harsh climate condi- 
tions, and also to restrictions included in the Antarctic legislation (as stated in the 
1959 Antarctic Treaty and its Protocol on Environmental Protection signed in Madrid 
in 1991, which prevent the introduction of any kind of nonindigenous organisms, 
including foreign micro-organisms for use in bioremediation), cleanup processes 
must be carried out using autochthonous bacterial strains. For this reason, a deep 
knowledge of the metabolic capabilities and the taxonomic position of the Antarctic 
micro-organisms able to proliferate in hydrocarbon-contaminated Antarctic soils is 
needed. Studies have been initiated only recently along those lines, with the result 
that little is known about the environmental impact of past human activities in the 
Antarctic, starting from the first settlements up to the 1980s. 

The aim of this chapter is to review current information on the presence of 
hydrocarbons in Antarctic soils and on their effect on the composition of the natural 
bacterial flora. Also discussed is the available knowledge on functions of the natural 
Antarctic bacterial flora and the effectiveness of bioremediation strategies based on 
the application of external innocula to hydrocarbon-contaminated Antarctic soils. 
References are made to similar works carried out in other studied extremely cold 
soils under hydrocarbon contamination. 



12.2 An Overview of the Bacteriology of Pristine 
Antarctic Soils 

A review of the Antarctic bacteriology (as for the bacteriology of any other site) 
requires that recent developments in bacterial taxonomy be taken into account. In 
addition, soil represents an extraordinarily complex environment, so that samples 
taken from different sites differ in their physicochemical properties including water 
and organic matter content, pH, presence of plant cover, presence of guano (which 
is typical of ornithogenic soils), level of nutrients, and many others. Climate 
conditions (such as temperature range, winds, and solar radiation regime) vary 
considerably among sites and even for a given site at different times. All these 
differences make it difficult to compare richness, abundance, and other soil biodi- 
versity determinants as they occur in different soil samples. As was remarked by 
Young and Crawford (2004), soil biology (including soil microbiology), soil chemistry, 
and soil physics are currently progressing almost as independent fields, which impedes 
the construction of an integrated view of soil ecology. Despite these limitations, 
a number of studies have examined the composition of the bacterial flora from 
extremely cold soils. 



250 L.A.M. Ruberto et al. 

Antarctic and Arctic soils are generally comprised of an active layer (exposed to 
a broad thermal oscillation ranging between +15°C and -35 °C) and a subjacent 
permafrost, which is defined here as the soil and/or rocks remaining below 0°C for 
at least two consecutive years (ACGR 1988). Permafrost is divided in an upper 
zone (0.5-20-m thick) with moderate temperature oscillations and a deeper perma- 
frost, showing a stable temperature regime (-5°C to -10°C). The Arctic and most 
of the Antarctica permafrost is ice-cemented. Exceptions are represented by the 
inland valley floors and sides of the McMurdo Dry Valleys, where the low water 
content determines the prevalence of dry (non ice-cemented) permafrost (Bockheim 
2002). Readers interested in a thorough description of the Antarctic active layer and 
permafrost are referred to the article by Bockheim and Hall (2002). In the present 
chapter, discussions are restricted to the bacterial communities colonizing the upper 
part of the active layer. The microbial ecology and biodiversity of permafrost has 
been extensively reviewed (Steven et al. 2003; see also Chapter 7). 

The active layer of the Antarctic soils is eminently aerobic and, consequently, 
strict anaerobes are infrequent. On the contrary, peatlands and other anaerobic habitats 
are frequent in the Arctic, offering a wide anaerobic niche for growth of strictly 
anaerobic bacteria (see Chapter 9). However, the presence of strict anaerobes 
(Clostridium and Eubacterium) in Antarctic soils has been reported as far back as 
55 years ago by Prevot and Moreau (1952). Several other Clostridium species were 
found in soils from the Antarctic Showa Station by Miwa (1975). More recently, 
other anaerobic bacteria were isolated from different Antarctic soils. Sulfate-reducing 
genera (Desulfovibrio and Desulfotomaculum) were identified from the peatlands 
of Signy Island (Christie 1987). In yet other studies on Antarctic permafrost and 
peats, anaerobic bacteria were detected by culturing (Brambilla et al. 2000) and 
several others were inferred using culture-independent methods (Brambilla et al. 
2000; Christner et al. 2003). However, extensive phylogenetic studies of Dry Valley 
mineral soils mentioned in a review by Cowan and Ah Tow (2004) gave no signals 
of 16S rRNA sequences from anaerobic groups. In the Antarctic active layer, where 
soil hydrocarbon contamination occurs, the presence of anaerobic bacteria is rare 
and the aerobic bacteria represent almost the total fraction of the bacterial flora. 

Although anaerobic pathways of hydrocarbon degradation exist (Heider et al. 
1998; Shinoda et al. 2005) and could be of relevance in subsurface environments 
such as the permafrost table or other anaerobic contaminated terrestrial Antarctic 
habitats, such processes have not been studied at the present time in nature and no 
information about the natural interactions between hydrocarbons and strictly 
anaerobic bacteria is currently available. Thus, for the most part the present review 
concentrates on the aerobic soil bacteria. 

An important number of bacterial isolates from Antarctic soils have been 
identified at the species level using biochemical and morphological tests. Several 
of these studies, carried out on vegetated soils, reported a significant fraction of 
coryneform bacteria. More than 30 years ago, Baker and Smith (1972) isolated species 
belonging to the genus Arthrobacter, Brevibacterium, Corynebacterium, Kurthia, 
and Cellulomonas from vegetated soils at Signy Island. The authors concluded that 
the microbial communities of the studied Antarctic soils were qualitatively and 



1 2 B acteriology of Hydrocarbon-Contaminated Cold Soils 25 1 

quantitatively similar to the microbiota of cold peat soils from other locations. 
Other members of the phylum Actinobacteria were frequently isolated from 
Antarctic soils using culture-dependent methods (Smith et al. 2000). Among 
Firmicutes, Bacillus spp. were widely reported (Johnson et al. 1978; Vishniac 1993), 
whereas Pseudomonas, Flavobacterium, and Agrobacterium were some of the most 
abundantly isolated Gracilicutes (Vishniac 1993; Zdanowski and Wqglenski 2001; 
Smith et al. 2006). An important diversity of cyanobacteria was also reported in the 
terrestrial environments (Nienow and Friedmann 1993), mainly in the well-studied 
Dry Valleys, as these bacteria were recognized as major components of the endo- 
lithic communities. Various other bacterial species have been occasionally reported 
in a great number of publications dealing with the isolation and identification of 
soil bacteria from different Antarctic areas. Readers interested in a detailed listing 
of the culture-dependent isolates from Antarctic soils are referred to the reports of 
Hirsch et al. (1985), Line (1988), Vincent (1988), and Vishniac (1993). 

Several works based on culture-dependent methods suggested that, at least in vege- 
tated soils, most Antarctic bacteria belong to a small number of cosmopolitan genera 
(Vishniac 1993). However, this assumption probably was biased by the fact that those 
common taxa comprise a high number of strains easily culturable in simple laboratory 
culture media. Many other important taxa of Antarctic soil bacteria, being, if not 
endemic to the Antarctic soils, at least exclusive to extremely cold environments, 
remain undetectable by the classical culture-dependent methods. These include fastidi- 
ous micro-organisms, and others that would be co-culture-dependent or that would 
occur in a viable but nonculturable state. The bias associated with culture-dependent 
methods was discussed by several authors (Franzmann 1996; Smith et al. 2006). 

Most of the novel micro-organisms described in the last 15 years using the new 
molecular techniques belong to genera that are not exclusive to the Antarctica. As 
the number of studies on bacterial composition of natural environments increased, 
it became evident that most of the genera described for a particular Antarctic envi- 
ronment (soils, ice, seawater, marine sediments) also had representatives in similar 
but non- Antarctic extremely cold environments (such as the Arctic or deep-sea 
environments). Thus, adaptation to cold (psychrophilia and psychro tolerance), 
rather than geographic location, seems to be the main determinant of the distribu- 
tion of the majority of the described genera. Finally, it must be noted that before 
1988, until molecular techniques were used, no member of the Archaea domain had 
been recognized in Antarctic environments (Franzmann 1996). 

Most of the Antarctic studies using molecular techniques have focused on 
bacterial communities from aquatic environments, particularly seawater, marine 
animals-associated bacteria, sea ice, and marine and lacustrine sediments. These 
studies led to the description of a great number of new Antarctic species (Bowman 
et al. 1997a,b; Bozal et al. 1997; Denner et al. 2001). On the other hand, fewer 
studies have dealt with Antarctic soil bacterial communities, so that our knowledge 
of the diversity of the nonculturable bacteria in these soils remains fragmentary. 
Unpublished data mentioned by Cowan and Ah Tow (2004) indicated that as many 
as 50% of the sequences retrieved from Miers Valley (Ross Desert) mineral soils 
are from as yet uncultured bacteria, whereas the remaining sequences belonged, 



252 L.A.M. Ruberto et al. 

either to taxa previously reported as being dominant by culture-dependent methods 
(27% of retrieved sequences were related to Actinobacteria and 11% to 
Bacteroidetes), or to other taxa with few cultivated representatives (6% of the 
sequences were related to Acidobacteria and 6% to Verrucomicrobia). 

When a lichen-dominated lithic bacterial community of the McMurdo Dry 
Valleys was studied using DNA-based methods (de la Torre et al. 2003) members 
of the order Cytophagales (a subgroup of the Bacteroidetes lineage) dominated. 
Other phylotypes, including Actinobacteria (Rhodococcus sp., Microsphaera sp., 
Blastococcus sp., Sporichthya sp.), Alphaproteobacteria (Sphingomonas sp.), 
Gammaproteobacteria (Actinobacter sp.), and members of Planctomycetales were 
detected in low proportion. In a recent publication (Aislabie et al. 2006b), 728 
clones from soil samples taken from four locations along 77 °S in Victoria Land, 
Antarctica, were characterized by restriction fragment length polymorphism 
(RFLP) of the 16S rDNA. DNA sequencing showed that the ribotypes occurring 
more than three times grouped within the bacterial divisions Bacteroidetes, 
Actinobacteria, Proteobacteria, Thermus/Deinococcus, Acidobacteria, Firmicutes, 
and Cyanobacteria. A salient feature of this study was the observed dominance of 
a few ribotypes occurring with an abundance of 10% of the analyzed clones or 
more, which represents an unusually high value for surface soils. However, the 
dominant ribotypes changed among locations. 

The presence of such over-dominant ribotypes could be a characteristic of the 
Antarctic soils (de la Torre et al. 2003) that is not frequently observed in soils from 
other latitudes, including cold soils from the Arctic (Zhou et al. 1997) or Colorado 
Alpine soils (Lipson and Schmidt 2004). These differences in community composi- 
tion may reflect adaptations to the diverse physicochemical and biological factors 
present in each studied soil and mainly to the water content (Aislabie et al. 2006b). 
Thus, bacterial groups such as Deinococcus and Rubrobacter only prevailed in the 
driest sampling site (Wright Valley) due to their desiccation tolerance. 

Analysis of microbial diversity from Arctic and Alpine tundra using 16S rRNA 
gene clone library sequencing methods showed that several of the detected bacterial 
groups were widely dispersed, occurring in these and also Antarctic soils. However, 
the relative abundance of such groups varied widely depending on the site and even 
on the season. Recently, Nemergut et al. (2005) have reviewed the data about 
Alpine and Arctic soil microbial communities, trying to relate this variability with 
ecological aspects of the studied systems. 



12.3 Hydrocarbons in Polar Soils 

The introduction of hydrocarbons in the Antarctic region results from a combina- 
tion of a global input flow from low-level and long-term natural and anthropogenic 
sources and accidental spills. Hydrocarbons can be introduced in the Antarctic envi- 
ronment from local natural sources such as lichens, algae, and bacteria (Neff 1979; 
Cripps 1989, 1990) and even meteorites have been reported as sources of Antarctic 



12 Bacteriology of Hydrocarbon-Contaminated Cold Soils 



253 



poly cyclic aromatic hydrocarbons (PAHs; Naraoka et al. 2000), although this has 
little relevance on the level of poly cyclic aromatic hydrocarbons in Antarctic soils. 
However, over this baseline of hydrocarbons from biogenic sources, localized con- 
tamination events with both aliphatic and aromatic hydrocarbons, have occurred since 
the onset of human activity in the Antarctic continent in the early 20 th century (Piatt 
and Mackie 1980). Humans generate hydrocarbon pollution mainly through the 
scientific stations, and through the logistic operations supporting the pelagic fisheries 
and tourism. The latter, in particular, has increased rapidly starting in the mid- 
1980s, and has now become an important activity (Bauer 2001). In fact, the recent 
grounding of the Norwegian cruise ship Nordkapp, which occurred in February 
2007 and caused a fuel spill in Deception Island, is a recent example of the pollu- 
tion risks related to this growing activity. 

Hydrocarbon contamination in the Arctic is a more frequent and extended 
problem than it is in Antarctica and has been more extensively studied. Very 
high levels of both aliphatic and aromatic hydrocarbons have been reported for 
Arctic soils (Mohn et al. 2001; Whyte et al. 2001). Alpine cold soils areas have 
also been reported as being affected by hydrocarbon pollution (Margesin and 
Schinner 2001). 

Which are the current levels of hydrocarbon contamination in Antarctic soils? 
The main hydrocarbon source in Antarctic soils is represented by fuels (crude and 
fuel oils) spilled on land during the environmentally risky activities of storage-tank 
filling and refueling of vehicles (Fig. 12.1). Several authors have reported high 




Fig. 12.1 Refueling activities in Antarctic Stations require transport of fuels from logistic ships 
to storage tanks by boat, helicopter, and/or pipeline. These operations are essential but imply an 
important environmental risk 



254 



L.A.M. Ruberto et al. 



aliphatic hydrocarbons levels caused by the mentioned activities (Gore et al. 1999; 
Delille 2000; Delille and Pelletier 2002). The aromatic fraction of these fuels is pre- 
dominantly represented by naphthyl (2-ring) compounds and the level of PAHs with 
3 or more rings (Cripps 1989) is low. However, these higher molecular- weight PAHs 
are the result of combustion processes of organic compounds. 

Although the Antarctic Treaty and its Protocol on Environmental Protection now 
restrict the incineration of wastes, for decades this was a frequent practice at the stations. 
It generated localized highly polluted areas rich in PAHs with 3 or more rings, which 
were generally associated with power generators and incinerators. For example, 
Kennicutt II et al. (1992a) reported that subtidal sediments below an abandoned open 
incineration site contained combustion-derived polynuclear aromatic hydrocarbons. 
Soils collected at Old Palmer Station were also contaminated with these kinds of 
compounds. Again in a recent study (Vodopivez et al. 2007, in press), carried out at 
Jubany Station (King George Island, South Shetland Islands), it was found that, 
although all sampling sites showed very low levels of PAHs, higher values were 
detected in combustion-related sites (boathouse, incinerator site). However, samples 
from one site where a diesel oil spill occurred did not show any increase of PAHs 
level compared with the control site. Table 12.1 shows some of the ^z-alkanes and 
PAHs levels reported from soils and sediments near Antarctic stations. 



Table 12.1 Total /i-alkanes and total PAH concentration in soils and coastal sediments reported 
for Antarctic areas 











PAHs in 










w-Alkanes in 


Surface 






/i-Alkanes in 


PAHs in 


Surface Marine 


Marine 






Surface Soil 


Surface Soil 


Sediments 


Sediments 




Sampling Area 


(ng/g dw a ) 


(ng/g dw) 


(ng/g dw) 


(ng/g dw) 


Reference 


King Edward 


nr b 


nr 


399 


16 


Mackie et al. 


Cove, South 










1978 


Georgia 












Islands 












Signy Island 


1,220,000 


71,000 


1,731 


280 


Cripps 1992 


Arthur Harbor 


2,344,971 


85,659 


121,851 c 


14,491 b 


Kennicutt II 
et al. 1992a 


Old Palmer 


1,182,092 


345,765 


772,734 


59,487 


Kennicutt II 


Station 










et al. 1992b 


Admiralty Bay 


nr 


nr 


nr 


32 


Bicego et al. 1998 


McMurdo 


nr 


88,452 


nr 


nr 


Mazzera et al. 


Station 










1999 


Scott Base 


nr 


8,105 


nr 


nr 


Aislabie et al. 
1999 


Admiralty Bay 


nr 


nr 


nr 


271 


Martins et al. 2004 


Potter Cove 


nr 


1,182 


nr 


1,908 


Curtosi et al. 
2007 



1 dw, dry weight. 
3 nr, not reported. 
; Intertidal sediment. 



12 Bacteriology of Hydrocarbon-Contaminated Cold Soils 255 

Several monitoring s of the Argentinean Antarctic Stations with permanent 
human presence have shown no significant problems of generalized contamination. 
However, as is the case for many stations from other countries, local manipulation 
of hydrocarbons has determined small restricted areas of pollution (Mac Cormack 
and Fraile 1997; Curtosi et al. 2007; Vodopivez et al. 2007). 

The fate of hydrocarbons accumulated in any polluted soil can vary greatly 
depending on several factors, including the chemical characteristics of the hydro- 
carbons (Lundstedt 2003), ageing (Alexander 2000; Dictor et al. 2003), the soil 
properties (Chiou et al. 1998; da Conceigao et al. 2006), the extent of the abiotic 
and biotic processes that promote their elimination from the contaminated site 
(Johnsen et al. 2005), and the enormously complex interactions which occur 
between them. Although an exhaustive analysis of each of the factors affecting the 
fate of hydrocarbons in soils exceeds the scope of this chapter, it is important to 
mention two of these factors that are characteristic of extremely cold soils. 

The first factor is the low temperatures existing in these soils, that slow down natu- 
ral biological processes. Micro-organisms show low specific affinity values (a A ) under 
polar temperatures, and thus become increasingly unable to sequester the substrates 
from their environment (Nedwell 1999). Under these conditions, even small quantities 
of hydrocarbons being spilled over a long time period could lead to a significant 
accumulation of these compounds due to the slow rate of natural biological degrada- 
tion. The second determining factor is the continuous process of freezing and thawing 
that is maintained in high latitude areas, which results in the smallest soil particles 
being selectively transported to deeper layers, whereas the upper layers become 
enriched in larger size particles (Anderson et al. 1978). This process favors downward 
migration of those hydrocarbons showing the highest affinity for the smallest soil par- 
ticles and modifies the distribution of the different hydrocarbons in the soil layers. 

In addition, the permafrost layer could act as a low permeability barrier for the 
migration of hydrocarbons. Biggar et al. (1998) have reported that, under certain condi- 
tions, some contaminants can penetrate the Canadian Arctic permafrost layer, and that 
this was a site-specific phenomenon. However, recent studies (Curtosi et al. 2007) about 
distribution of PAHs in soils near Jubany Station showed the existence of a concentra- 
tion gradient of PAHs, with levels being relatively low at the soil surface and increasing 
with depth in the active layer, reaching a maximum just below the interface between the 
active layer and the permafrost, and then progressively declining with depth in the per- 
mafrost (Fig. 12.2). These results show that both the freezing and thawing cycles and 
the permafrost barrier exert a significant effect on the distribution and fate of hydrocar- 
bons in polar soils. Accompanying effects will be observed on the distribution of the 
bacterial flora able to tolerate (or degrade) such hydrocarbons. 

Table 12.1 presents chronic hydrocarbon contamination events, where soils have 
been exposed to the pollutant for long time periods. A very different situation is cre- 
ated by acute contamination events, where significant amounts of hydrocarbons are 
spilled on a previously pristine soil. In this case, the effect produced on the microbi- 
ota, which was not adapted to the presence of contaminants, is very different from 
that caused by a chronic contamination. From the above discussion on factors 
influencing hydrocarbon mobility and other soil characteristics, it remains difficult to 



256 



L.A.M. Ruberto et al. 



PAHs concentration (ng/g) 

20 40 60 



80 



50 ■ 



^ 100- 



150- 



200 



250- 



300 




Fig. 12.2 Total PAHs concentrations in the active layer and permafrost from a sampling site near 
Jubany Station (South Shetland Islands, Antarctica). Samples were taken in February 2005 and 
patterns similar to that shown here were observed in several other sites near the station 



predict the effect hydrocarbons will have on the natural bacterial flora. From here on, 
we present several studies dealing with the bacterial flora of hydrocarbon-contami- 
nated Antarctic soils. This is done with the intention to extract, beyond the specifici- 
ties of each particular case study, some general conclusions on the response of the 
bacterial communities to the presence of hydrocarbons. Similar studies performed in 
non- Antarctic extremely cold soils are mentioned in order to analyze whether similar 
responses can occur in all those soil communities under similar stress situations. 



12.4 Bacteriology of Hydrocarbon-Contaminated Polar Soils 



Studies on the bacteriology of hydrocarbon-contaminated Antarctic soils have been 
impelled mainly by the intention to apply the best hydrocarbon-adapted micro-organisms 
to the development of bioremediation processes of contaminated Antarctic and 
other extremely cold soils. Different studies have dealt with the analysis of aerobic 
heterotrophic (AHB) and hydrocarbon-degrading (HDB) bacterial populations, 
using either culture-dependent or culture-independent methods (Mac Cormack and 
Fraile 1997; Baraniecki et al. 2002; Delille et al. 2003; Ruberto et al. 2003; Saul 
et al. 2005). 

It is important to distinguish the hydrocarbon-contaminated soils as a function 
of the acute or chronic nature of the contamination event. One possible situation is 



12 Bacteriology of Hydrocarbon-Contaminated Cold Soils 257 

that of the pristine soil, without previous exposure to hydrocarbons, which sud- 
denly receives a spill of a fuel such as diesel oil or jet aircraft fuel (JP1). This situa- 
tion results in an "acutely contaminated soil". On the other hand, there are soils 
receiving small but repetitive doses of hydrocarbons. In this case, the prevailing 
hydrocarbons may be mainly aliphatic, aromatic, or a complex mixture of these, 
and the soil remains exposed to the pollutants for periods of years or decades. These 
soils are "chronically contaminated". From a microbiological point of view, bacteria 
from an acutely contaminated soil are naive with respect to hydrocarbon degrada- 
tion, whereas those from a chronically contaminated soil have become adapted to 
the pollutant. 

In general, Antarctic soils are acutely contaminated by petroleum-derived fuels, 
resulting in an early decrease in total bacterial counts as well as in a loss of diversity. 
This effect is probably caused by a toxicity effect of the hydrocarbons exerted on 
the nontolerant members of the bacterial community. After this acute deleterious 
effect of a hydrocarbon spill, evolution of population density of culturable AHB 
may take different courses. After crude oil contamination of nine sub- Antarctic 
intertidal beaches, Delille and Delille (2000) found no relevant changes in the AHB 
population density during a 90-day long experiment. On the contrary, when a con- 
trol and contaminated soil samples from Scott Base were compared (Saul et al. 
2005), AHB counts in contaminated soils were one to two orders of magnitude 
higher than in the control soil. A significant increase in AHB was also observed in 
a pristine Antarctic soil contaminated with diesel oil 28 days after contamination in a 
microcosm assay (Ruberto et al. 2005). In contrast with these observed differences 
in the evolution of AHB population density, enrichment of HDB in soils following 
acute contamination is general. Indeed, with respect to HDB population size, incre- 
ments of several orders of magnitude were reported following acute contamination, 
resulting in the relative fraction of HDB among the general population rising from 
almost negligible levels to relatively high values. Several assays carried out by our 
and other research groups have shown that hydrocarbon-degrading bacteria are 
present even in pristine Antarctic soils and that their initially low numbers rise sig- 
nificantly after contamination. 

Some Antarctic pristine soils showed numbers of hydrocarbon degraders 
extremely low or even under detection limits (Aislabie et al. 1998, 2001). However, 
hydrocarbon degraders represented an initial proportion of 3.2% of total AHB 
counts in pristine surface soils from Jubany station (Ruberto et al. 2003), and this 
proportion rose to 80-100% 28 days after contamination with gas-oil. Similarly, 
hydrocarbon degraders amounted to 0.1% of AHB counts in a 75 cm deep soil 
showing low levels (1.2ppm) of PAHs (Ruberto et al. 2006), but this proportion 
increased to 100% 56 days after phenanthrene contamination. Also the number of 
HDB reported by Delille et al. (2003) for pristine soils in the vicinity of Dumond 
d'Urville station (Terre Adelie), which represented less than 2% of the total AHB 
counts, increased by several orders of magnitude after diesel and crude oil 
contamination. 

The above-cited studies differed according to the characteristics of the soils 
examined and methods for evaluation of both AHB and HDB numbers. For example, 



258 L.A.M. Ruberto et al. 

in some studies the Most Probable Number technique was used, whereas others 
resorted to plate-count methods on various culture media and carbon sources. Such 
differences preclude close comparisons between the various studies. However, the 
fact remains that HDB are present in all the studied Antarctic soils, even in those 
with no previous exposure to a detectable contamination from anthropogenic origin, 
and that their numbers significantly increase after an acute contamination event. 
This conclusion applies to other cold soils, including Alpine (Margesin and 
Schinner 1997b) and Arctic (Thomassin-Lacroix et al. 2002) soils. The only known 
exception to this observation with respect to cold soils comes from analysis of soils 
near Scott Base, where a reduced number of AHB and an undetectable level 
of hydrocarbon degraders were attributed to the presence of very high (and toxic) 
levels of lead from leaded fuels (Aislabie et al. 1998). 

Little is known about changes in bacterial diversity of Antarctic pristine soils 
after hydrocarbon pollution. Studying the culturable fraction of bacterial commu- 
nity and using biochemical techniques for identification of the isolates, Ruberto et al. 
(2003) found that, upon hydrocarbon contamination, a pristine soil bacterial 
community possessing members of Agrobacterium, Pseudomonas, Acinetobacter, 
Moraxella, Flavobacterium, Bacillus, Micrococcus, Xanthomonas, Entero- 
bacteriaceae, and unidentified Gram-negative cocci was reduced to a "contaminated 
community" where only Pseudomonas and Acinetobacter members were detected. 
Although it considered only the culturable microbial fraction, this study suggested 
that bacterial communities from pristine Antarctic soils may suffer a significant 
reduction in diversity following exposure to hydrocarbons. 

In a more recent study, Saul et al. (2005) found that control soils from Scott Base 
were dominated by Fibrobacter/Acidobacterium (20%), Actinobacteria (17%), 
Bacteroidetes (10%), Proteobacteria (6%), Thermus/Deinococcus (3%), and low-GC 
Gram positive bacteria (2%), this conclusion being based on the culture-independent 
analysis of 155 clones. However, hydrocarbon-contaminated soils, from which 367 
clones were analyzed, were significantly less diverse, Proteobacteria (76%) being 
by far the dominant division. Although these authors found important differences 
when the bacterial community of the control soils was analyzed using culture- 
dependent methods (no members of the Fibrobacter/Acidobacterium division were 
recognized by culturing), cultural isolations from contaminated soils yielded similar 
results to those obtained using culture-independent methods, with Proteobacteria, 
mainly represented by isolates from the genera Pseudomonas, Sphingomonas and 
Variovorax, as the dominant group and accounting for 65% of the total AHB 
population. 

When chronically contaminated Antarctic soils were studied, they showed a low 
bacterial diversity and a high proportion of hydrocarbon-degrading bacteria, an 
effect similar to that observed upon acute hydrocarbon contamination of pristine 
soils. Several hydrocarbon-degrading bacterial strains were isolated from chronically 
contaminated Antarctic soils. These strains belong to a number of genera comprising 
Rhodococcus (Bej et al. 2000; Ruberto et al. 2005), Acinetobacter (Mac Cormack 
and Fraile 1997), Pseudomonas (Panicker et al. 2002), Sphingomonas (Baraniecki 
et al. 2002), Shewanella (Gentile et al. 2003), and others. Rhodococcus seem to be 



12 Bacteriology of Hydrocarbon-Contaminated Cold Soils 259 

dominant in Antarctic soils where aliphatic hydrocarbons are the main contaminants 
but not in PAHs-contaminated Antarctic soils. Rhodococcus species were predomi- 
nant among the alkane-degrading isolates from Scott Base (Bej et al. 2000), where 
C9-C14 chain lengths were the most abundant rc-alkanes. These Rhodococcus 
strains grew on a broad range of rc-alkanes (from C6 to C20) as well as on the 
branched alkane pristane. No growth was observed when PAHs (toluene and naph- 
thalene) or cyclohexane were present as the sole carbon source. 

Similar results were reported by Ruberto et al. (2005) following examination of 
Jubany Station soils under diesel and JP fuels contamination. In this case, 
Rhodococcus strains grew on C10-C16 chain lengths rc-alkanes and several alkane- 
rich fuels but failed to grow on xylene, pyrene, and phenanthrene. Only one of the 
three studied Rhodococcus strains grew on cyclohexane, and showed an important 
surfactant activity associated to the cell surface (Fig. 12.3). Yet another relevant 
characteristic of the above-mentioned strains, shared with the bulk of the 
hydrocarbon-degrading Antarctic strains isolated from soils, was their psychrotol- 
erance (but no psychrophily). Although the three Rhodococcus strains can grow at 
0-5° C, optimal growth temperature ranged from 20-30°C. This psychro tolerance 
property seems to be a general feature of hydrocarbon-degrading bacteria isolated 
from the surface of cold soils, where the temperature can drop to -30°C or less 
during winter, whereas in summer long-term solar exposure can raise the soil 




0.1 \im 



Fig. 12.3 Electron micrograph of Antarctic Rhodococcus ADH (Ruberto et al 2003). This strain 
has been isolated from a fuel-contaminated soil near Jubany Station and, as several other Antarctic 
hydrocarbon-degrading Rhodococcus strains, can use crude oil, a number of light fuels, and a 
broad range of alkanes as a carbon source. When grown on hydrocarbons, strain ADH has an 
important surfactant activity and frequently shows membrane complexes (as can be observed in 
the lower part of the image), probably associated with hydrocarbon metabolism 



260 L.A.M. Ruberto et al. 

temperature to 20°C or more (Margesin and Schinner 1991a; Whyte et al. 1997; 
Deppe et al. 2005). Under these conditions, psychrotolerant bacteria possess a crucial 
adaptive advantage over the strict psychrophiles. The prevalence of Rhodococcus 
and other Actinobacteria in fuel-contaminated cold soils has also been confirmed 
by culture-independent methods in the case of Arctic soils (Juck et al. 2000), but 
not of Alpine soils (Margesin et al. 2003). 

A search for alk genes has shown that homologues of two alkane hydroxylase 
systems are present in contaminated as well as uncontaminated Antarctic soils 
(Whyte et al. 2002), suggesting that alkane-metabolizing members of the 
Rhodococcus genus are common inhabitants of both pristine and hydrocarbon- 
contaminated soils. It seems that Rhodococcus represents one of the main com- 
ponents of the HDB in pristine Antarctic soils and that the spill of alkane-rich 
fuels generates a selective pressure that allows members of this genus to prevail 
in a chronic-contamination situation. Another genus that was reported as a relevant 
group in some pristine soils is Acinetobacter (Ruberto et al. 2003). Although 
some species of this genus were isolated from fuel-contaminated Antarctic soils 
(Mac Cormack and Fraile 1997), their prevalence in Antarctic contaminated sites 
seems to be low (Whyte et al. 2002). On the contrary, Acinetobacter sp. was 
reported to be significantly enriched from contaminated Alpine soils (Margesin 
et al. 2003). 

The metabolic versatility of Pseudomonas and related organisms from the 
Gammaproteobacteria is well known (Palleroni 1995), with various representatives 
exhibiting the capacity for utilization of a wide range of organic compounds as the 
carbon and energy source, including aliphatic and aromatic hydrocarbons. As was 
mentioned in Section 12.2, Pseudomonas has been frequently found in Antarctic 
(as well as in Arctic and Alpine) pristine soils. Although their ability to metabolize 
aliphatic hydrocarbons in low -temperature environments is well documented 
(Stallwood et al. 2005), it is in PAHs-contaminated environments, including cold 
soils, that Pseudomonas and related genera clearly became dominant. Isolates of 
these genera have frequently been reported to use PAHs as a substrate (Whyte et 
al. 1997; Aislabie et al. 2000). Through phylogenetic analysis, these studies also 
showed that Pseudomonas strains isolated from Antarctica and the Arctic were 
closely related and fell in the same cluster. 

Recently, the composition of the Antarctic bacterial consortium M10, isolated 
from a contaminated Antarctic soil by enrichment cultures on PAHs (phenanthrene, 
anthracene, fluorene, and dibenzothiophene) was determined (Mestre 2006). All 
the culturable strains characterized using 16S rDNA sequencing and biochemical 
techniques were Pseudomonas, Stenotrophomonas, and Burkholderia. In addition 
to this culture-based analysis, direct cloning of total DNA from M10 consortium 
followed by 16S rDNA sequencing was performed, with very similar results except 
for the detection of a minor member of the Bacteroidetes. 

A similar phylogenetic study recently performed by Ma et al. (2006) with 22 
PAH-degrading bacterial strains isolated from Antarctic soils with naphthalene 
and phenanthrene as the sole carbon source resulted in the identification of 21 
Pseudomonas and one Rahnella sp., again pointing to the important role played 



12 Bacteriology of Hydrocarbon-Contaminated Cold Soils 261 

by Pseudomonas in PAHs-contaminated cold environments. As the ndo (naphtha- 
lene dioxigenase) genes of the Antarctic Pseudomonas showed no differences 
with ndo genes from mesophilic strains, it was proposed that these genes were 
acquired by the Antarctic Pseudomonas through horizontal gene transfer from 
exogenous strains. Margesin et al. (2003), who also observed a sharp increase in 
the percentage of genotypes containing degradative genes similar to those of 
Pseudomonas putida in contaminated Alpine soils compared to pristine controls, 
attributed this increase to the r-type strategy followed by pseudomonads, which 
are fast colonizers and grow rapidly on nutrient-rich materials, such as hydrocar- 
bons. This being the case, it may be that at a later stage of the contamination 
process, when the readily degraded compounds are consumed, other bacterial 
groups, more adapted to low nutrient concentrations and showing a K-type strat- 
egy (such as Rhodococcus species), occupy this ecological niche and replace 
Pseudomonas and other fast-growing bacteria. 



12.5 Bioremediation in Extremely Cold Soils 

The existence of natural hydrocarbon-degrading bacteria in pristine Antarctic soils 
and their increase in numbers after exposure to these contaminants suggest that 
bioremediation may help reduce hydrocarbon contamination in these and other cold 
soils. As polar areas are remote and transferring the affected soils to more temper- 
ate areas is very expensive and frequently impracticable, the treatment of affected 
soils on site (i.e., on or near the site where the contamination event occurs) seems 
to be the best option. 

As was mentioned above (see Section 12.1), bioremediation techniques for 
cleaning hydrocarbon-contaminated soils have been successfully applied to 
the cleaning of temperate soils all around the world. Although extremely cold soils 
might not be an exception, a number of factors, and primarily low temperatures, 
tend to limit microbial activity in these environments. Antarctic micro-organisms 
are adapted to survive and grow, not merely at low temperatures, but also in the 
presence of temperature oscillations and the consequent freeze-thaw cycles resulting 
from temperatures rising well above 0°C during the short summer period (see 
Section 12.4). For example, the soil at Jubany station, located in the North part of 
the Antarctic Peninsula (where a large part of the Antarctic stations are estab- 
lished), can reach 15-20°C during a sunny day in the summer, but the temperature 
can then drop to values below 0°C at night. A strictly psychrophilic micro-organism 
could not survive these temperature fluctuations, whereas psychro tolerant strains 
harbor an adaptive advantage under these particular conditions. 

Although the prevailing low temperatures determine a low bacterial metabolic 
rate and reduce evaporation of volatile compounds, a significant abiotic loss of 
hydrocarbons has been reported when fuels are spilled on Antarctic soils. Ruberto 
et al. (2003) reported that at least 54% of an initial level of 14,380 ppm from an 
acutely diesel-contaminated soil was lost in 10 days due to volatilization and 



262 L.A.M. Ruberto et al. 

stripping. A lesser but relevant abiotic elimination of diesel oil (16-23%) was found 
by Margesin and Schinner (1997a) during a laboratory assay where an acute spill 
was simulated on Alpine soils. However, despite the abiotic loss of the light com- 
ponents of fuels suddenly spilled on soils, a significant fraction of the hydrocarbons 
remain in the soil many years after the contamination event (Gore et al. 1999). This 
fact suggests that, in many cases, biodegradation rate of the natural Antarctic 
microbiota is too low to eliminate the contaminants. 

An unequal spatial distribution of micro-organisms and contaminants and a retar- 
dation of substrate diffusion in the soil matrix can strongly limit hydrocarbon bioa- 
vailability (Harms and Bosma 1997). Enell et al. (2005) have shown this retardation 
process (particularly relevant with PAHs) to be temperature-dependent and found 
that the desorption rate of several PAHs from contaminated soil decreased by a factor 
of 11-12 when temperature declined from 23 to 7°C. Thus, the low temperatures at 
which the extremely cold soils are exposed could exacerbate the substrate diffusion 
limitation. The extent of biodegradation also depends on the chemical structure 
(Heitkamp and Cerniglia 1987) and the physical state of the compounds (Wodzinski 
and Coyle 1974). Several other factors limiting biodegradation of hydrocarbons in 
polar soils, such as pH, nutrient availability, and soil moisture, were reviewed in a 
recent and complete paper by Aislabie et al. (2006a). 

There is no general consensus about the best strategy for bioremediation of 
Antarctic (and others extremely cold) soils. Although natural attenuation proved to 
reduce hydrocarbon levels in recently contaminated sub-Antarctic (Delille and 
Pelletier 2002) and Alpine soils (Margesin and Schinner 2001), the time required 
for this process and the remaining levels of hydrocarbons suggest that other strate- 
gies such as bio stimulation and bioaugmentation represent valid alternatives to 
improve the speed and extent of the biodegradation in Antarctic soils. 

It seems that chronically contaminated areas, where natural microbiota is adapted 
to the hydrocarbons, need no additional bacterial inocula for a successful biodegra- 
dation. A chronically contaminated soil (containing 1 2,000 ppm of diesel and JP1 
fuels) from Marambio Station was dispensed in separate 3-kg quantities and placed 
in metal trays (Fig. 12.4a) to compare the effects of biostimulation of the autoch- 
thonous microbiota with inorganic N and P sources and of bioaugmentation with 
different hydrocarbon-degrading bacterial strains and consortia (Ruberto et al. 
2004). After 45 days, the biostimulated autochthonous bacterial flora was as effi- 
cient for hydrocarbon degradation as the best bioaugmented system. We have 
obtained similar results working with chronically contaminated soils from Jubany 
Station and using 1 m 2 land plots as an experimental model (Fig. 12.4b). The same 
conclusion was obtained by other researchers working in the Arctic (Whyte et al. 
1999; Thomassin-Lacroix et al. 2002). In the Alps, Margesin and Schinner (1997b) 
found that bioaugmentation of diesel oil-contaminated soils enhanced biodegradation 
rates only slightly and temporarily. Hence, bioaugmentation of chronically contami- 
nated cold soils would serve only to reduce the length of the initial acclimation period 
by the natural microbiota, and would not increase the extent of the biodegradation 
processes. In any given situation, an exhaustive time-cost balance analysis should 
be made to determine the best bioremediation strategy. 



12 Bacteriology of Hydrocarbon-Contaminated Cold Soils 



263 




Fig. 12.4 Different microcosm systems used as experimental models in studies on bioremedia- 
tion of Antarctic soils, (a) Metallic trays with 3 kg of soil; (b) 1 m 2 land plots; (c) small flasks with 
20 g of soil; (d) larger flasks with 250 g of soil 



The possibility remains that the relatively low impact of bioaugmentation is due 
to what is called by Thompson et al. (2005) "the challenge of the strain selection." 
These authors argue that selection of strains has been frequently done on the basis 
of their catabolic competence but not according to many other essential features 
that are crucial for the function and persistence of the biological model in the target 
habitat. In this sense, recent results using molecular techniques to detect the pres- 
ence and progress of two bacterial consortia used for bioaugmentation of Antarctic 
soils (Vazquez et al. 2007), suggest that consortia do not survive the prevailing 
environmental conditions and are outcompeted by the natural microbiota, when 
they are inoculated at densities similar to those shown by the culturable fraction of 
the soil community (10 6 -10 7 colony-forming units (CFU)/g dry soil). Further stud- 
ies using higher inoculum levels (10 9 CFU/g or more) must be done to determine 
to what extent the inoculation of chronically contaminated soils improves the 
hydrocarbon degradation activity. An infrequent case where inocula had significant 
stimulatory effects on bioremediation in field experiments was reported by Mohn 
et al. (2001), using biopiles for on-site bioremediation of chronically hydrocarbon- 
contaminated tundra soils. This stimulatory effect of bioaugmentation was signifi- 
cant during the first summer after treatment (39-53 days), but no difference with 
the fertilized systems was observed one year after treatment. 

A very different situation arises upon contamination of a previously pristine soil. 
In this case, the low numbers of hydrocarbon-degrading bacteria suggest the need 



264 L.A.M. Ruberto et al. 

for inoculation (bioaugmentation) with previously isolated hydrocarbon-degrading 
bacteria. However, contradictory results have been obtained in such systems. 
A significant improvement in degradation efficiency was obtained when an 
Antarctic soil acutely contaminated with diesel oil was inoculated with indigenous 
hydrocarbon-degrading bacteria (Ruberto et al. 2005). A positive effect of bioaug- 
mentation was also obtained when a soil showing low levels (1.2ppm) of PAHs was 
exposed to l,744ppm of phenanthrene (Ruberto et al. 2006) in the presence offish 
meal (as N and P source) and a surfactant (Brij®700) as a bioavailability enhancer. 
Bioaugmentation with a Pseudomonas strain (Stallwood et al. 2005) also enhanced 
the bioremediation rate in an uncontaminated Antarctic soil exposed to 1% w/w 
Polar Blend marine diesel oil. 

All of the above-mentioned studies used microcosms, performed in flasks filled 
with small quantities of soil, as an experimental model (see Fig. 12.4c,d). Although 
few studies have been conducted along those lines, the currently available literature 
on artificially contaminated Antarctic soils indicates that bioaugmentation could be 
a valid strategy when a fuel is accidentally spilled on pristine soils. Under this situ- 
ation, inoculation could effectively reduce the lag period intervening before the 
HDB counts rise and, thus, improve the extent of pollutant elimination. This accel- 
eration of bioremediation processes would be highly desirable in Antarctica and the 
Arctic, where a significant biodegradation activity is only possible during the short 
summer period. 

Whereas the usefulness of bioaugmentation strategies still remains controversial, 
the requirement of N and P addition seems obvious for Antarctic soils, where the levels 
of these nutrients are generally low. Although it is true that many hydrocarbon- 
degrading bacteria are well adapted to oligotrophic conditions prevailing in soils 
(Johnsen et al. 2005), the presence of high levels of carbon source (represented by 
the pollutants) leads to an unbalanced C:N:P ratio which limits bacterial growth. As 
a fraction of the hydrocarbons is used to generate bacterial biomass, additional lev- 
els of N and P are required to support this biomass increase in polluted soils. 

As was mentioned above (see Section 12.4), a surge in bacterial growth and 
activity is often observed in the first stages of hydrocarbon biodegradation proc- 
esses in cold soils. In these cases, both the natural bacterial flora and the bacterial 
inocula used for bioaugmentation require additional amounts of N and P. Some 
studies with Antarctic soils used commercial slow-release fertilizers, such as Inipol 
EAP-22, for biostimulation. This product, which is a microemulsion containing 
nitrogen (urea) and phosphorus (tri(laureth-4) -phosphate) encapsulated within oleic 
acid, efficiently enhanced biodegradation of alkanes in soils from Kerguelen 
Islands when added at a C:N:P ratio of 100:12:1.1. However, no effect of the ferti- 
lizer on the PAHs degradation rate was detected (Delille et al. 2004). In addition, 
the stimulating effect of the fertilizer was stronger on a desert soil having low levels 
of nutrients than on a vegetated soil. When used for bioremediation of an Alpine 
soil, a commercial water-soluble N-P-K fertilizer enhanced biodegradation of diesel 
oil at an N:P ratio of 20:1 (Margesin and Schinner 2001). Biostimulation was also 
effective when N and P were added as inorganic salts ( (NH 4 ) 2 S0 4 and K 2 HP0 4 ) on 



12 Bacteriology of Hydrocarbon-Contaminated Cold Soils 265 

flask-microcosms prepared with oil-contaminated Antarctic soils from South 
Orkney Islands (Stallwood et al. 2005). The effectiveness of bio stimulation was 
also demonstrated with Arctic soils using diverse nutrient sources as urea and diam- 
monium phosphate (Thomas sin-Lacroix et al. 2002) or a water-soluble fertilizer 
(Braddock et al. 1997). 

However, some results highlight the care that must be taken when N and P are 
added as "enhancers" of the bioremediation processes. Ruberto et al (2003) found 
that when N and P were added to diesel oil-contaminated Antarctic soils as inor- 
ganic salts (NaN0 3 and Na 2 HP0 4 ) at a C:N:P ratio of 100:12:3, an initial inhibition 
of bacterial growth, and hence a slower hydrocarbon degradation activity, was 
observed. It is possible that the relatively high amounts of nutrients used in this 
study (l,800mg N/kg soil and 500 mg P/kg soil) were inhibitory to the microbiota. 
This hypothesis is in keeping with the results obtained by Braddock et al. (1997) 
who found that 50-100 mg N/kg enhanced microbial hydrocarbon degradation in 
soils from Alaska, whereas 200 mg N/kg were inhibitory. Negative effects of 
(NH 4 )N0 3 and K 2 HP0 4 were also observed in non-cold soils by Trindade et al. 
(2002) at C:N ratios of 100:10 and 100:5 and a C:P ratio of 100:5. 

Soil texture strongly influences the optimum level of N for hydrocarbon deg- 
radation. Indeed, sandy soils (which represent the majority of Antarctic soils and 
have a lower water-holding capacity than the silt- and clay-rich soils), are more 
prone to inhibition by inorganic nitrogen (Walworth and Reynolds 1995). Also, 
depending on their structure, hydrocarbons might require different levels of nutri- 
ents for their degradation. This relationship was suggested by the observation by 
Breedveld and Sparrevik (2000) that 3 -ring and 4-ring PAHs were degraded to 
different extents in Norwegian cold soils. Finally, a comparison of available 
results from various studies suggests that what might be considered as a "high 
level" of nutrients depends on a number of factors, including the tolerance of the 
microbiota involved, the physical characteristics of the soil, and the type of N and 
P source used. 

For these reasons, it is difficult to define the adequate level of nutrients 
required by a soil bacterial community for an optimum degradation activity. 
If only the C:N:P ratio is taken into account, the highly contaminated soils pre- 
senting levels as high as 1 0,000 ppm of TPH or more would require the addition 
of N and P to levels that could be toxic or inhibitory for the microbiota, if the aim 
were to reach a C:N:P ratio of 100:10:1. In recent years, the use of complex 
organic materials as slow-release nutrient sources has been successfully tested. 
Biostimulation of the autochthonous microbiota using fish meal improved 
phenanthrene removal from Jubany Station soils (Ruberto et al. 2006). Dry fish 
compost also was reported by Pelletier et al. (2004), working with soils from the 
Kerguelen Archipelago at the northern limit of the Antarctic ocean, as an efficient 
fertilizer as well as an excellent solid carrier for bioremediation additives 
(nutrients and surfactants). The use of such low-cost complex organic matrixes is 
also being explored in several other regions for bioremediation of temperate soils 
(Rahman et al. 2002; Molina-Barahona et al. 2004). 



266 L.A.M. Ruberto et al. 

12.6 Major Challenges for the Near Future 

One of the major challenges for the near future is not related exclusively to the cold 
soils or to their exposure to the contaminants. It is a challenge inherent to the com- 
plexity of the soil matrix in general. As stated in Section 12.2, at the present time 
there exists no theory linking the dynamics of the bacterial communities to biodi- 
versity and function in terms of the different microenvironments existing in the soil 
(Young and Crawford 2004). Progress along those lines will require the combined 
efforts of ecologists, microbiologists, molecular biologists, soil physicists, and 
mathematicians, to fully account for the complexity of the problem. Of course, such 
multidisciplinary effort would also benefit our understanding of the bacterial com- 
munities of the cold soils as well as their functioning and evolution upon pollutant 
exposure. 

As regards hydrocarbons in cold soils, in spite of the valuable contributions of 
several research groups, many important aspects remain poorly studied, especially 
with respect to Antarctic soils. Although much information has been gathered from 
other areas, little is known about the effect of hydrocarbons of different chemical 
compositions on the Antarctic soil ecosystems, either in the short or in the long 
term. Due to the characteristics of Antarctic food webs, with scarce terrestrial pri- 
mary producers, low diversity of macroorganisms, and seasonal migration of most 
consumers located at the highest trophic levels to more temperate regions, Antarctic 
ecosystems are particularly sensitive to perturbation. Hence, alteration of the resi- 
dent bacterial community by hydrocarbons could have enormous effects on the 
function of the sensitive Antarctic ecosystems, these effects occurring up to the 
highest trophic levels. 

Several questions remain unanswered in relation to the fate of hydrocarbons in 
extremely cold soils. The presence of the permafrost introduces a unique feature 
that changes the distribution and migration of hydrocarbons in an unknown way. 
Recent results obtained by us in Jubany Station (Curtosi et al. 2007; see also Section 
12.3) showed that hydrocarbons accumulate in the permafrost table. Conclusions 
from these studies on the fate of hydrocarbons in extremely cold soils should be 
validated for many other sites in Antarctica and the Arctic by applying well- 
planned monitoring programs. It will lead us to estimate the consequences of the 
global warming that probably will affect the structure of permafrost. If the perma- 
frost table, retaining a significant concentration of PAHs, is melted and the com- 
pounds washed out to the near marine basin, a massive flow of PAHs may ensue, 
with unpredictable ecological consequences. 

We have only scant knowledge of the metabolism of the different hydrocarbon 
compounds in cold soils. Because different micro-organisms can metabolize the 
same hydrocarbon compound using different pathways and producing different 
metabolites (Sutherland et al. 1995), deeper studies on the biochemical fate of 
hydrocarbons in nature should be conducted. The issue is more than strictly 
academic, as some metabolites can result in being highly toxic to the natural biota. 
For this reason, the potential of the soil microbiota to produce any particular 



12 Bacteriology of Hydrocarbon-Contaminated Cold Soils 267 

intermediate compound bears relevance on the foreseeable progress of hydrocarbon 
degradation and the fate of the individual components spilled on the soil. 

In relation to the bioaugmentation strategies, one of the main limiting steps is 
the lack of practical tools for investigating the fate of the bacterial inoculum. This 
problem is made more complicated still when a consortium (and not a single bacte- 
rium) is used and when the consortium has been obtained from the treated location. 
Because allochthonous micro-organisms cannot be used for bioremediation in 
Antarctica, to monitor the fate of the inoculated micro-organisms throughout the 
processes represents a real challenge. It will be necessary to find solutions to this 
problem before bioaugmentation can be considered as a valuable alternative. 
Related unsolved problems concern the immobilization of the inoculum and strategies 
for inoculum application to soils. Immobilization has been analyzed recently in 
order to extend the permanence in the soil of the inoculated hydrocarbon-degrading 
bacteria and hence to enhance removal of the contaminants (Cunningham et al. 
2004). However, these studies were carried out at a laboratory scale and not using 
extremely cold soils. Thus, there is currently no information about in situ bioaug- 
mentation of cold soils with immobilized bacteria. 

Inoculation strategies that improve hydrocarbon removal are needed for the 
Antarctic soils. It has been reported that repeated inoculation enhances hydrocarbon 
mineralization rates (Schwartz and Scow 2001). However, studies using Antarctic 
soil micro-organisms exposed to harsh climatic conditions are needed to define the 
adequate inoculation strategy for in situ bioremediation involving bioaugmentation. 
Progress along those lines would contribute to address the problem pointed out by 
Aislabie et al. (2004): to date, no consensus has been reached on remediation 
guidelines for hydrocarbon contamination cleanup protocols for the Antarctic. 

12.7 Conclusions 

Extremely cold soils have a natural microbiota composed mainly of psychro tolerant 
micro-organisms adapted to survive under the prevailing extreme environmental 
conditions. Additional stress represented by the presence of hydrocarbons of anthro- 
pogenic origin tends to increase the heterotrophic bacterial counts and markedly 
stimulate the hydrocarbon-degrading bacterial fraction. However, contaminated 
soils present lesser bacterial richness and diversity in comparison to pristine 
controls. Currently, bioremediation strategies are considered as one of the best 
alternatives to restore the restricted Antarctic areas where hydrocarbons represent 
a real contamination problem. As was observed in other areas of the world, bios- 
timulation of the natural bacterial flora seems to be the adequate choice for reducing 
the level of hydrocarbons from chronically contaminated cold soils. 

On the other hand, bioaugmentation currently represents a controversial strategy 
that seems to be useful only in soil with no previous exposure to the pollutants or 
with low populations of hydrocarbon-degrading bacteria. For Antarctica, as human 
activities (particularly scientific research, fisheries, and tourism) increase, new and 
deep multidisciplinary knowledge is needed to face the problem of hydrocarbon 



268 L.A.M. Ruberto et al. 

contamination and to realize the goals stated by the Antarctic treaty and its Protocol; 
in particular, it is intended that Antarctica shall continue forever to be used exclu- 
sively for peaceful purposes and that its status as a special conservation area will 
be preserved. 



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Chapter 13 

Microbiology of Oil-Contaminated Desert Soils 

and Coastal Areas in the Arabian Gulf Region 



Samir Radwan 



13.1 Introduction 

Deserts are of global distribution; they cover considerable areas of all continents, 
with the exception of Europe. Desert soils are poor in organic substances and water, 
and are usually subjected to rather high temperature in summer and chilling in 
winter, and to extensive light. In spite of their extreme character, desert soils usually 
accommodate communities of micro-organisms including actinomycetes, cyano- 
bacteria and other bacteria, fungi, protozoa, and phototrophic microalgae. Many of 
such micro-organisms live naturally under stress, and must possess special adaptive 
mechanisms in order to survive and propagate (see Chapter 2). Desert micro-organisms 
appear to be limited in their physiological activities due to low availability of certain 
nutrients, according to Liebig's "law of the minimum" (Liebig 1840). 

Sometimes, microbial activities in the desert soil are arrested according to 
Shelford's "law of tolerance" (Shelford 1913) saying that there are maxima and 
minima for environmental factors above and below which micro-organisms cannot 
survive. Nutrient starvation seems to be one of the most serious problems desert 
soil micro-organisms have to overcome. Apparently, primary producers such as 
microalgae and cyanobacteria are the major sources of organic materials in the poor 
desert soils. Roszak and Colwell (1987) identified among bacteria a number of sur- 
vival approaches against starvation, that fall within two strategies (Jannasch 1967), 
namely the potential for growth at low nutrient levels, and the potential for entering 
into dormancy. 

Another possible mechanism for survival of starving bacteria is cell size reduc- 
tion through the phenomenon of multiple division, thus producing the so-called 
ultramicrobacteria (Novitsky and Morita 1976, 1977, 1978; Morita 1982). The 
smaller the bacterial cell, the larger is its surface-to-volume ratio, and consequently 
the greater is its potential for accumulating diluted nutrients from the surroundings. 



Samir Radwan 

Department of Biological Sciences, Kuwait University, P.O. Box 5969, Safat 13060, Kuwait 

e-mail: radwan @kuc01. kuniv.edu. kw 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 275 

© Springer- Verlag Berlin Heidelberg 2008 



276 S. Radwan 

Ultramicrobacteria are widely distributed in marine waters (Button et al. 1993). 
They may be rather dormant and consequently, relatively resistant to environmental 
stresses (Roszak and Colwell 1987). Some starving bacteria with a depleted amino 
acid pool exhibit the so-called "stringent response" (Neidhardt et al. 1990) which 
ultimately reduces the protein synthesis rate by inhibiting rRNA synthesis. 

In addition to stresses exerted on desert micro-organisms by factors such as nutrient 
deficiency, drought, and heat, additional stress may arise due to pollution of the same 
desert soil areas with crude oil. This is particularly true for oil-producing countries 
such as the Arabian Gulf countries. Such desert soil areas have been polluted for 
many ages with small amounts of hydrocarbon vapors naturally volatilizing from the 
deep oil reservoirs. However, the pollution problems associated with modern and 
intensive oil production and transport occur on a much expanded scale. 

In addition, coastal regions of oil-producing countries are particularly exposed 
to oil pollution which of course exerts stress on the indigenous coastal microflora. 

The objective of this chapter is to shed light on changes in the indigenous 
microflora of desert and coastal regions in response to pollution. Emphasis is put 
on the desert and coastal regions of the Arabian Gulf for several reasons. Oil- 
utilizing micro-organisms of the Gulf area have now been the subject of study in 
our laboratory for more than 15 years. This area contains ancient oil, and has pro- 
duced oil intensively for decades. The Gulf oil spill associated with and resulting 
from the Iraqi occupation of Kuwait, from August 2, 1990, to February 26, 1991, 
is so far the greatest in the history of mankind. 

13.2 The Gulf Oil Spill 

Shortly before their withdrawal from Kuwait in February 1991, the Iraqi forces 
deliberately blew up the Kuwaiti wells, amounting to more than 700 in the desert. 
It took the Kuwaiti authorities about seven months to get the resulting fires under 
control. During that period, crude oil kept gushing, and thus about 300 so-called 
"oil lakes" (Fig. 13.1) formed, covering in total about 50km 2 of the Kuwaiti Desert. 
There are estimates (McKinnon and Vine 1991) that such lakes used to contain 
about 22 million barrels of oil, but 18 million barrels have been recovered and 
exported, and 3 million remain as pollutants. Oil penetrated between 40 to 60 cm 
deep into the sand. The total volume of polluted desert soil is estimated to be 20 x 
10 6 m 3 , and still contains highly viscous to solid crude. 

The Gulf water body also received a share of the oil pollution. The Iraqi forces on 
January 19, 1990 deliberately released crude oil from the Mina Al-Ahmady oil terminal 
directly into the water. According to different estimates, the amount of oil released in 
the course of three successive days ranged between half a million and twelve million 
barrels (McKinnon and Vine 1991). The slick was 16-km long, that is, several times 
the size of the famous Exxon Valdez spill in Alaska. Most of the oil was transported 
counterclockwise by the water currents to the south. Most of the crude then became 
sedimented in the intertidal zone along more than 700 km of the western Gulf coast, 
leaving the water and the subtidal zone almost free of oil sediments. 



13 Microbiology of Oil-Contaminated Desert Soils 



277 





Fig. 13.1 One of the small oil lakes in the Kuwaiti desert (February 1993) 

13.3 Composition of Crude Oil 

Crude oil consists chemically of four major constituents: saturates, aromatics, 
asphaltenes, and resins (Leahy and Colwell 1990). Saturated hydrocarbons, including 
normal alkanes with chains of up to 44 carbon atoms, branched alkanes, and 
cycloalkanes (naphtenes), are the major constituents of the crude, making up 
between 40 and 60% of the total weight. Aromatic hydrocarbons with from one to 
six benzene or substituted benzene rings follow the saturates in quantitative impor- 
tance and amount to roughly 20% of the crude weight. Asphaltenes, which include 
tar, are very high-molecular weight hydrocarbons, which are used as road paving 
materials. Resins are crude constituents that contain, in addition to carbon and 
hydrogen, sulfur and oxygen. The chemistry of asphaltenes and resins is still not 
yet completely known; both constituents make up 1 to 5% of light oils and up to 
25% of heavy oils, which correspondingly contain lower proportions of saturates 
and aromatics. 



13.4 General Description of Oil-Utilizing Micro-Organisms 



Several reports have been published on oil-utilizing micro-organisms, for example, 
Klug and Markovetz (1971), Levi et al. (1979), Einsele (1983), Radwan and 
Sorkhoh (1993), Van Hamme et al. (2003), Rosenberg (2006), and Widdel et al. 
(2006). It is important to note that such micro-organisms are normal indigenous soil 



278 S. Radwan 

and water inhabitants, and that most of them can consume conventional carbon 
sources. Their defining characteristic, which makes them capable of utilizing 
hydrocarbons as substrate, is that they possess the so-called mono-oxygenase and/ 
or dioxygenase enzyme systems. Such systems catalyze the introduction of oxygen 
atoms from molecular oxygen into aliphatic and aromatic hydrocarbon molecules 
producing the corresponding alcohols that in turn become further oxidized to 
aldehydes, and ultimately acids. The resulting acids are then biodegraded by 
p-oxidation, producing acetyl CoA that can be further metabolized (for reviews see 
Rehm and Reiff 1981; Fukui and Tanaka 1981; Boulton and Ratledge 1984). 

The capacity to utilize hydrocarbons is widely distributed among conventional 
micro-organisms including prokaryotes and eukaryotes. Bacterial genera reported to 
attack hydrocarbons include Acinetobacter, Micrococcus, Vibrio, Azospirillum (Roy 
et al. 1988), Aeromonas, Alcaligenes, Chromobacterium, Flavobacterium, Klebsiella, 
Pseudomonas (Klug and Markovetz 1971), Bacillus (Loginova et al. 1981; Sorkhoh 
et al. 1993), Arthrobacter, Brevibacterium, Corynebacterium, Rhodococcus, 
Mycobacterium, Nocardia and other nocardioforms (Egorov et al. 1986), and 
Streptomyces (Barabas et al. 1995). Yeast genera capable of utilizing hydrocarbons 
include (for review see Radwan and Sorkhoh 1993) Candida, Dabayomyces, 
Endomyces, Leucosporidium, Lodderomyces, Metschnikowia, Pichia, Rhodo- 
sporidium, Rhodotorula, Saccharomycopsis, Schwanniomyces, Selenotila, 
Sporidiobalus, Sporobolomyces, Torulopsis, Trichosporon, and Wingea. Filamentous 
fungi with hydrocarbon utilization potential include Absidia (Hoffman and Rehm 
1978), Aspergillus, Aureobasidium, Beauveria (Davies and Westlake 1979), Botrytis, 
Cephalosporium, Cladosporium, Corellospora, Canninghamella, Dendyphiella 
(Kirk and Gordon 1988), Fusarium, Hormodendrum (Lin et al. 1971a,b), Lulworthia 
(Kirk and Gordon 1988), Mortierella, Mucor, Penicillium, Phialophora, Phoma 
(Davies and Westlake, 1979), Scedosporium (Ornodera et al. 1989), Scoleobasidium 
(Davies and Westlake 1979), Sporotrichum, Varicosporina, and Verticillium (Kirk 
and Gordon 1988). In addition, there are reports that phototrophic bacteria such as 
Rhodo spirillum and Rhodopseudomonas (Cerniglia et al. 1980b); cyanobacteria such 
as Oscillatoria (Cerniglia et al. 1980a), Microcoleus, and Phormidium (Al-Hasan 
et al. 1994, 1998); microalgae such as Chlamydomonas and Chlorella (Ellis 1977); 
and the phytoflagellate Euglena (Ellis 1977) can oxidize aliphatic and/or aromatic 
hydrocarbons. 

None of the micro-organisms listed above can consume all of the crude oil con- 
stituents; and each organism has the potential for utilization of only a limited range 
of compounds. Yet, collectively, all crude constituents from the gaseous low molecular 
weight (van Ginkel et al. 1987; Ornodera et al. 1989) up to the medium and high 
molecular weight (Demanova et al. 1980) compounds, including asphaltenes, can 
be attacked by micro-organisms. 

Growth on oil and hydrocarbons is associated in some micro-organisms with 
certain unique morphological and/or cytological features. One of the most frequent 
features is the appearance of cytoplasmic hydrocarbon inclusions in actinomycetes 
(Barabas et al. 1995) and other bacteria (e.g., Scott and Finnerty 1966; Atlas and 
Heintz 1973; Kennedy and Finnerty 1975), and also in filamentous fungi (Cundell 



13 Microbiology of Oil-Contaminated Desert Soils 279 

et al. 1976; Koval and Redchitz 1978; Redchitz 1980). The picocyanobacteria 
Synechococcus and Synechocystis exhibit much wider interthylakoid spaces in 
the presence of oil and hydrocarbons than in the absence of these compounds 
(Al-Hasan et al. 2001). Some micro-organisms produce dense intraplasmic membranes 
(Kennedy and Finnerty 1975; Ivshina et al. 1982) and volutin inclusions (Redchitz 
and Koval 1979; Ivshina et al. 1982). Penicillium grows in shaken cultures in the 
presence of hydrocarbons as hollow mycelial balls enclosing hydrocarbon droplets, 
whereas in hydrocarbon-free media the balls are solid (Cundell et al. 1976). 



13.5 Oil-Utilizing Micro-Organisms in the Arabian 
Gulf Desert Soils 

Our group in Kuwait, working for more than 15 years on oil and hydrocarbon- 
utilizing micro-organisms indigenous to the desert and marine environments of the 
Arabian Gulf, has collected a wealth of useful information on this subject. This 
information may help in understanding the composition of oil-utilizing microflora 
indigenous to other desert and coastal areas similar to those of the Gulf area. 

The predominant indigenous oil-utilizing bacteria in the Kuwaiti desert belong 
to Micrococcus, Pseudomonas, Bacillus, Arthrobacter, and the group of nocardio- 
forms, particularly the genus Rhodococcus (Sorkhoh et al. 1990, 1995; Radwan et 
al. 1997). The genus Streptomyces is the predominant oil-utilizing actinomycete in 
the Kuwaiti desert (Barabas et al. 1995, 2000; Radwan et al. 1998b). 

The oil-utilizing fungal flora of the Kuwaiti desert comprises predominantly the 
genera Aspergillus, Penicillium, Fusarium, and Mucor. Members of other genera 
are also found (Sorkhoh et al. 1990). The Gulf region is characterized by a rather 
long, dry, and very hot summer. Therefore, it may be expected that the desert in this 
region may accommodate thermophilic hydrocarbon-utilizing micro-organisms. 
The analysis of 38 Kuwaiti Desert soil samples polluted with crude oil revealed the 
occurrence of 3.7 x 10 3 to 1.1 x 10 7 cells of thermophilic (with an optimal tempera- 
ture of 55°C) oil-utilizing bacteria per g of soil, all of which were identified as 
Bacillus stearothermophilus (now Geobacillus stearothermophilus) (Sorkhoh et al. 
1993). The isolation of hydrocarbon-utilizing thermophiles is not unexpected 
inasmuch as Loginova et al. (1981) observed the growth of obligate thermophilic 
bacteria in a medium with paraffin. Similarly, Zarilla and Perry (1984) reported on 
Thermoleophilum album as a novel bacterium obligate for thermophily and utilizing 
n-alkane substrates. 

In the course of our studies on oil-utilizing micro-organisms in the Kuwaiti 
Desert soils, we noticed that oil-polluted areas generally contained higher numbers 
of such organisms than pristine areas. However, pristine desert soil was never free of 
oil-utilizing bacteria. In particular, in both pristine and contaminated Kuwaiti Desert 
areas, the soil fraction in direct contact with the desert plant roots (or rhizo sphere) 
represented microenvironments enriched in oil-utilizing micro-organisms. The 
rhizospheres of desert plants growing in the Kuwaiti Desert were found to contain 



280 S. Radwan 

more hydrocarbon-utilizing micro-organisms than the soil farther away from the 
roots (Radwan et al. 1995b, 1998a). These plants included Senecio glaucus, Cyperus 
cenglomeratus, Launaea mucronata, Picris babylonica, and Salsola imbricata. It 
was observed that some plants, although growing in black, oil-polluted, desert soil 
areas, possessed white clean roots rich in oil-utilizing bacteria (Radwan et al. 
1995b). Not only the rhizospheres of wild plants, but also those of legume crops 
(e.g., Vicia faba and Lupinus albus) were richer in hydrocarbon-utilizing bacteria 
than nonrhizo sphere soil. The rhizosphere effect (which is measured as the ratio 
of the number of micro-organisms in the rhizosphere soil to the number of 
micro-organisms in nonrhizosphere soil (Anderson et al. 1993)) was much more 
pronounced for plants growing in oil-polluted than in pristine soils. The most 
prevalent hydrocarbon-utilizing bacteria in the rhizospheres of the above plants were 
Cellulomonas flavigena, Rhodococcus erythropolis, and Arthrobacter spp. 

There is experimental evidence for self-cleaning of oily desert soil in Kuwait through 
the activities of the indigenous hydrocarbon-utilizing microflora. The total amounts of 
extractable alkanes from heavily polluted soil cores in a Kuwaiti oil field exposed to the 
open air were quantitatively determined once every two weeks through a whole year 
(Radwan et al. 1995a). It was found that the total amount of extractable alkanes 
remained fairly constant during the dry hot summer months, but decreased during the 
rainy months reaching, after one year, slightly more than one half of the amount at 
zero time. This result demonstrates the self-cleaning capacity of the Kuwaiti Desert soil 
and the essential role of moisture in this process. The loss of alkanes could not be 
attributed to simple physical volatilization, because loss occurred at a slower pace 
during the hot summer. On the contrary, self-cleaning was faster during the rainy period 
of the year, suggesting that it was mainly occurring through biological processes. 



13.6 Micro-Organisms in the Gulf Coastal Areas 

One of the interesting observations our group made during trips to the oily coastal 
regions in the early 1990s was the appearance of mats of an intense blue-green 
color at the top of oil sediments in the Saudi Arabian Gulf coasts, about 300 km 
south of Kuwait (Sorkhoh et al. 1992). Those mats are frequent in the Gulf coasts, 
even in the nonpolluted areas (Golubic 1992). Later, we made similar observations 
along the oily Kuwaiti coasts (Fig. 13.2). Strikingly, the mats were tightly associ- 
ated with oil, and oil-free coastal areas were also free of those mats. It was also 
noted that all forms of higher life on the oily coasts were dramatically inhibited by 
the oil sediments. Animal inhabitants were absent or dead, and their coastal under- 
ground tunnels were full of crude. The mats appeared to be at that time the only, or 
one of the few living things in the oily coasts. The microbiological analysis of mat 
samples we collected from the Saudi research station of Jubail revealed that they 
consisted mainly of photo synthetic and heterotrophic prokaryotes. The phototrophs 
included filamentous cyanobacteria, such as Microcoleus, Phormidium, Spirulina, 
and others, in addition to some eucaryotes, mainly diatoms. The filamentous 



13 Microbiology of Oil-Contaminated Desert Soils 



281 







Fig. 13.2 Microbial mats on the top of oil sediments along the Kuwaiti Coast of the Arabian 
Gulf 



cyanobacteria adhered together through excreted mucilage forming the mat matrix. 
The heterotrophic bacteria associated with those mats included millions of oil- 
utilizing bacteria per g fresh mat. 

In this context, blue-green mats, also comprising the cyanobacterium Microcoleus, 
have also been recorded in the pristine coast of Abu Dhabi (Golubic 1992). The 
hydrocarbon-utilizing bacteria associated with the mats (Sorkhoh et al. 1995) consisted 



282 S. Radwan 

of nocardioforms (63%), belonging mostly to the genus Rhodococcus, the genera 
Bacillus (21%), and Arthrobacter (13%), in addition to Pseudomonas as a minor 
bacterium. Actinomycetes belonging to the genus Streptomyces and filamentous fungi 
belonging to the genera Aspergillus and Penicillium were also identified, but were 
much less frequent than the other heterotrophs. Microcoleus consortia comprising 
heterotrophic bacteria capable of biodegrading oil have also been described by Garcia 
De Oteyza et al. (2004). The microbial consortium in the blue-green mats is active in 
self-cleaning of the oily coasts of the Gulf. Successive visits to the lightly polluted 
coasts along Kuwait revealed that the oil sediments gradually vanished. Today, 
those coasts have become absolutely oil-free, and their mats have disappeared. 

The mats harbor a number of advantages as valuable biological systems in self- 
cleaning of oily coasts. Heterotrophic oil-utilizing bacteria are naturally immobilized 
within the mats, thus avoiding being washed out into the open sea. Furthermore, 
such bacteria are adequately aerated with oxygen produced by the photo synthetic 
partners in the mats. It is known that oxygen could be a limiting factor in microbial 
biodegradation of hydrocarbons (for a review, see Radwan and Sorkhoh 1993). In 
addition, some of the cyanobacterial partners in the mats are probably nitrogen fixers 
(see also Steppe et al. 1996); nitrogen fertilization is known to enhance microbial 
degradation of hydrocarbons (Radwan et al. 1995c). In addition, there is experi- 
mental evidence for direct hydrocarbon oxidation by cyanobacteria and algae 
(Cerniglia et al. 1980a,b; Al-Hasan et al. 1994, 1998; Raghukumar et al. 2001; 
Todd et al. 2002). 

Along the Gulf coast and also probably elsewhere, littoral materials from inter- 
tidal zones are associated with much higher numbers of oil-degrading micro-organisms 
than inshore and offshore water samples (Radwan et al. 1999). This may indicate 
that the coasts have a better potential for oil biodegradation than the water body. 
Oil-utilizing bacteria found associated with coastal materials along the Arabian 
Gulf belonged to the genera Acinetobacter, Micrococcus, and the group of nocar- 
dioforms (Radwan et al. 1999). Interestingly, those bacteria are more frequent in 
association with littoral animate materials, such as microbial mats and epilithic 
biomass, than in association with inanimate materials, such as sand, stonelets, and 
gravel particles. However, gravel particles coated with blue-green biofilms are also 
rich in oil-utilizing bacteria (Radwan and Al-Hasan 2001). 

In coastal and offshore waters of the Gulf, oil-utilizing bacteria are found prefer- 
entially associated with macroalgae (Radwan et al. 2002), fish (Radwan et al. 
2007a), and picoplankton (Radwan et al. 2005a), rather than free-living. Thus, 
microbial consortia in biofilms along the coasts and various associations in the water 
body seem to play a major role in self-cleaning of the oily marine ecosystems. 

13.7 Extremophilic Oil-Utilizing Micro-Organisms 

There is an information gap regarding the extremophilic oil-utilizing micro-organisms 
in desert, coastal, and marine environments. Mention has already been made of the 
few studies on thermophilic hydrocarbon-utilizing bacteria (Loginova et al. 1981 



13 Microbiology of Oil-Contaminated Desert Soils 283 

Zarilla and Perry 1984; Sorkhoh et al. 1990; see Section 13.5). However, there are 
no studies on hyperthermophilic hydrocarbon-utilizing bacteria. 

A significant number of oil-polluted ecosystems are characterized by rather high 
alkalinities and/or salinities. These include estuaries, beaches, salt marshes, inland 
lakes, rockpools, desert rain pools, and others. Furthermore, billions of gallons of 
wastewaters with high contents of salts and waste organics are generated by indus- 
try, and disposed of in the environment. An example of such industrial activities is 
the production, transport, and refining of crude oil, normally associated with the 
generation of a large volume of oily salt water that displays a wide range of alka- 
linities and/or salinities (Flynn et al. 1996; Roe et al. 1996). This makes oil pollu- 
tion difficult to treat using conventional microbial strains, whose cell membranes 
may be disrupted and enzymes denatured (Kargi and Dincer 2000; Woolard and 
Irvine 1994). To bioremediate oily alkaline and saline environments without costly 
pretreatment, alkaliphilic and halophilic micro-organisms should be used (Diaz 
et al. 2002). 

With these facts in mind, recent work performed in our laboratory (Sulaiman, 
2006; Al-Awadhi et al. 2007) concerned the isolation and identification of alkaliphilic 
and halophilic oil-utilizing bacteria from the Arabian Gulf coasts of Kuwait. The 
results showed that animate coastal materials such as epilithic biomass and cyanobacterial 
mats were associated with considerable numbers of alkaliphilic and halophilic oil- 
utilizing bacteria. Inanimate material, such as coastal sand and gravel particles, as 
well as coastal waters contained much fewer numbers, if any, of these bacteria. The 
alkaliphilic oil-utilizing bacteria were found to belong to the genera Marinobacter, 
Micrococcus, Dietzia, Bacillus, Oceanobacillus, and Citricoccus. The halophilic oil- 
utilizing bacteria were found to belong to the genera Marinobacter, Georgenia, 
Microbacterium, Stappia, Bacillus, Isoptericola, and Cellulomonas. All isolates had 
a good potential for hydrocarbon degradation, and consequently may be suitable tools 
for self-cleaning and bioremediation for oily alkaline and salty regions. 



13.8 Microbial Consortia and Associations 

In previous sections of this review, mention has been made of the frequent occur- 
rence of microbial associations probably involved in oil biodegradation in the Gulf 
environments (see also Grotzschel et al. 2002; Abed et al. 2002; Abed and Koster 
2005; Sanchez et al. 2005). Such associations were recorded in the desert and 
coastal environments as well as in the water body of the Gulf. In the present section, 
more light is shed on those associations. Although it should be expected that oil- 
biodegradation in oily desert soil is mediated by "hypothetical" microbial consortia 
comprising bacteria and fungi, with the possibility of establishment of cometabolic 
and syntrophic strategies, there is only little information on the role of individual 
partners in such consortia. 

The rhizospheric microflora associated with roots of desert plants may offer 
unique opportunities in this respect, as in this case the benefits of the association 



284 S. Radwan 

to the oil-utilizing microbes are obvious. In the rhizosphere, oil-utilizing 
micro-organisms find proper conditions for propagation and activity. These 
micro-organisms can cover their vitamin requirements from exudates excreted by 
the root tissues. Indeed, more than 90% of oil-utilizing bacteria need vitamins for 
optimal growth and activity (Radwan and Al-Muteirie 2001). Furthermore, plants 
are known to aerate their rhizospheres by pumping air down into the soil. Oxygen 
was reported to be a limiting factor for the microbial attack on hydrocarbon 
molecules (Rehm and Reiff 1981; Fukui and Tanaka 1981). The roots, particularly 
of legumes, may also provide oil-utilizing bacteria with nitrogen fixation potential, 
thus enriching soil with compounds reported to enhance microbial hydrocarbon 
biodegradation (Atlas 1981; Leahy and Colwell 1990). 

The coastal and aquatic marine environments appear to contain more interesting 
oil-degrading microbial consortia than those of the desert soil. In most of those 
associations there are phototrophic partners and heterotrophic oil-utilizing partners. 
This is true for coastal epilithic biomass (Radwan et.al. 1999), coastal cyanobacterial 
mats (Radwan et al. 1999), coastal gravel particles coated with picocyanobacteria 
and other phototrophs (Radwan and Al Hasan 2001), macroalgae coated with 
bacterial biofilms (Radwan et al. 2002), and picocyanobacteria associated with 
oil-utilizing bacteria at the water surface (Radwan et al. 2005a). In all these consortia, 
the conditions are suitable for growth and activity of oil-utilizing bacteria. Thus, 
oxygen becomes available as a byproduct of photosynthesis, and nitrogenous com- 
pounds as a result of nitrogen fixation by some cyanobacteria. Furthermore, as 
mentioned above, in many of these associations the bacteria are protected against 
dispersal in the open sea. 

Another interesting association in coastal and offshore waters is that of oil- 
utilizing bacteria in biofilms coating fish surfaces and gills and gut linings (Radwan 
et al. 2007a). All test samples of ten types of the Arabian Gulf fish and two of farm 
fish were found to accommodate rather high numbers of oil-utilizing bacteria, with 
10 5 to 10 7 cells being found per cm 2 of fish surface and per gram of gills and guts. 
Such numbers were much higher than in the surrounding Gulf water which 
contained only 10 2 — 10 3 bacteria per ml. Such bacteria belonged to the genera 
Acinetobacter, Micrococcus, Bacillus, Rhodococcus, and other nocardioforms. 
It should be expected that ships, boats, and other vehicles navigating in the Gulf 
(and other open waters) are also probably coated by biofilms rich in oil-utilizing 
micro-organisms which apparently play a role in self-cleaning of the polluted 
aquatic environments. 



13.9 Responses of Micro-Organisms to Environmental 
Variables 

The numbers and activities of oil-utilizing micro-organisms in oily desert soil and 
coastal regions are affected by a number of environmental factors. 



13 Microbiology of Oil-Contaminated Desert Soils 285 

13.9.1 Organic Matter Content 

There is a lack of information on the effect of desert soil organic matter on oil- 
utilizing micro-organisms. Desert soils are characterized by their very low content 
of organic substances. Therefore, it may be assumed that the organic oil pollutants 
would specifically favor oil-utilizing micro-organisms. They certainly do, and we have 
observed this through our studies on the Gulf desert microflora. How oil-utilizing 
micro-organisms would be affected, should an oily desert soil sample receive 
conventional organic substances, should also be examined. This question is rele- 
vant, because most of the oil-utilizing micro-organisms also have the potential for 
utilizing conventional organic carbon sources. A partial answer to this question lies 
in the fact that hydrocarbon-utilizing micro-organisms are more frequent in the 
rhizospheric than in the nonrhizo spheric soils (Radwan et al. 1998a). It is well 
established that plant roots permanently excrete into the soil organic exudates, for 
example, sugars, vitamins, and amino acids. Such exudates probably stimulate the 
oil-utilizing micro-organisms in the rhizospheric soils. 

A more direct answer to the interesting question of conventional substrate 
utilization is provided by the results of one of our studies (Radwan et al. 2000). 
It was found that fertilizing an oily sample of desert soil with a mixture of glucose 
and peptone resulted in enhanced oil attenuation in that sample. The magnitude of 
the stimulation effect was too great to be attributed solely to nitrogen fertilization 
by the added peptone. Soil fertilization with KN0 3 containing an equivalent 
amount of nitrogen to that in peptone brought about a much lower oil attenuation 
value than that obtained with peptone. Glucose/peptone addition to a clean desert 
soil sample resulted in a dramatic increase of the total numbers of oil-utilizing 
micro-organisms in that sample. After 13 days, the micro-organisms had depleted 
all the added glucose and peptone and their numbers decreased. In the oily desert 
soil sample, glucose/peptone addition also increased the numbers of oil-utilizing 
micro-organisms. Yet, after the depletion of glucose and peptone, the numbers of 
oil-utilizing micro-organisms remained high and enhanced oil attenuation was 
recorded. It was thus concluded that easily utilizable conventional carbon and 
nitrogen sources in desert soil favor the oil-utilizing microflora, and consequently 
oil attenuation in such soils. 

A question may be raised here regarding the origin of easily utilizable organic 
matter in the poor desert and coastal soils. It is well known that the primary producers 
in the various ecosystems of our planet are the phototrophic (and chemolitho- 
trophic) organisms. In this respect, coastal areas, being wet all or most of the time, 
benefit from supporting algae and cyanobacteria. The latter produce and liberate 
easily utilizable organic matter via photosynthesis. On the other hand, dry desert 
soils seem to support such phototrophs only temporarily, following rainy periods. 
Therefore, their organic substance contents should be low, and dependent on the 
frequency of precipitation. 

Certain organic compounds, such as carboxylic acids, alcohols, and aldehydes, 
inhibit or even kill most micro-organisms. Interestingly, hydrocarbon-utilizing 



286 S. Radwan 

micro-organisms permanently produce such compounds from their hydrocarbon 
substrates as metabolic intermediates and release some of them into the environ- 
ment (for a review see Radwan and Sorkhoh 1993). 



13.9.2 Temperature 

Micro-organisms in the Gulf desert soils are naturally subjected to a rather wide 
range of temperatures. During daytime the surface desert soil in the Arabian Gulf 
environment may reach or exceed 70°C. In winter nights the temperature may fall 
below the freezing point. Although there are a few reports in the literature on ther- 
mophilic hydrocarbon-utilizing bacteria (Loginova et al. 1981; Zarrilla and Perry 
1984; Sorkhoh et al. 1993), our experience indicates that hydrocarbon-utilizing 
micro-organisms in desert soils and coastal regions of the Gulf are predominantly 
mesophilic with optima at 30-35 °C. We have isolated only one thermophilic species, 
Bacillus stearothermophilus, from desert soils, and this organism was not markedly 
predominant in any of the numerous desert soil samples analyzed (Sorkhoh et al. 
1993). We also failed to isolate obligate psychrophilic hydrocarbon-utilizing 
micro-organisms from desert soil samples. 

It appears that desert micro-organisms are provided with survival mechanisms at 
both high and low temperatures. Our knowledge of such mechanisms is still far 
from clear. The production of "dormant" units such as endospores, cysts, and others, 
is one of such mechanisms; yet it is limited only to a few species of bacteria and 
fungi. However, diminishing metabolic activity in vegetative cells to a minimum 
level may probably be an effective strategy for survival at unsuitable temperatures. 
This strategy might be more easily applied at suboptimal than at superoptimal 
temperatures. The moisture content may be an important interfering factor here. 
Moist heat is known to be more effective in killing micro-organisms than dry heat. 
Dry proteins need higher temperatures for denaturation than wet proteins. In view 
of the fact that the highest temperatures in the desert environment are reached 
during the long "dry" summer, a preliminary understanding of probable surviving 
mechanisms of mesophilic micro-organisms at temperatures much above their 
optima could be gained through examination of dry heat effects on proteins. 

Wet coastal regions appear to be more protected against the high summer 
temperatures than the dry desert areas. 



13.9.3 Hydrogen Ion Concentration 

Our routine measurements revealed that pristine and oily desert soils of the Gulf 
were rather neutral. This was also true for coastal soil samples except for a tendency 
of these soils to become slightly alkaline. It is known that soil pH affects the disso- 
ciation of the carboxyl and amino groups of proteins and thus the microbial enzymatic 



13 Microbiology of Oil-Contaminated Desert Soils 287 

activities. For optimal activity, enzymes have to be in a certain state of dissociation. 
Furthermore, the pH affects the solubility and consequently the availability of many 
nutrients, for example, phosphate and ammonium (see Atlas and Bartha 1998). It 
has been mentioned (see Section 13.4) that hydrocarbon-utilizing micro-organisms 
in the Gulf desert soils and coastal areas comprise bacteria and fungi. Bacteria pre- 
dominantly prefer neutral environments and are usually sensitive to acidity. Most 
fungi also prefer a neutral pH value, but are tolerant of acidity. 

These facts mean that the microbiological attack on hydrocarbons in the Gulf 
desert soils and coastal areas can occur at a wide range of hydrogen ion concen- 
trations, yet with an optimum at pH 7. In view of the well-known fact that fungi 
are commonly slower in growth and activity than bacteria, it should be expected 
that acidity would slow down the hydrocarbon biodegradation in the polluted 
soils. On the other hand, the coastal areas of the Gulf contain limited numbers of 
alkaliphilic hydrocarbon-utilizing bacteria (Al-Awadhi et al. 2007). This also 
means that extremely alkaline areas polluted with oil could be enriched with 
alkaliphilic hydrocarbon-utilizing bacteria that would sustain the oil biodegrada- 
tion process. 

Hydrocarbon biodegradation in oily soils proceeds in the pH range of 4.5 to 1 1.5 
with optima between pH 6.5 and 8 (Daylan et al. 1990). Our group found that the 
irrigation of oil desert soil samples with sewage-effluent as source of water and 
nitrogen inhibited alkane-biodegradation due to increasing soil acidity (Radwan 
et al. 1995c). However, liming relieved this inhibition. 



13.9.4 Moisture and Aeration 

Desert soils are characterized by extremely low moisture content most of the year. 
In the Arabian Gulf region, precipitation is rather rare and occurs only during the 
short winter. The dry period may exceed nine months in the year. On the other hand, 
coastal areas are submerged with sea water during tidal and wave movements, and 
suffer from drought only temporarily. There is an inverse proportion between the 
soil moisture content and the soil degree of aeration. Thus, the Gulf desert soils are 
well aerated most of the time. Microbial activities in dry soils are substantially 
enhanced by increasing the water content, but only until the latter starts to fill the 
soil air space. When water logging occurs, growth and metabolism of aerobic 
micro-organisms become inhibited. Moisture content of soil is optimal for residing 
micro-organisms at 50-75% of its water holding capacity. 

Oil-utilizing micro-organisms are predominantly aerobic, and the first step in the 
microbial attack on hydrocarbon substrates involves the introduction of one (for 
alkanes) or two (for aromatic rings) oxygen atoms into the substrate molecule 
(Rehm and Reiff 1981; Boulton and Ratledge 1984; Buehler and Schindler 1984; 
Singer and Finnerty 1984). There are reports on anaerobic biodegradation of hydro- 
carbons, but such a process is so slow that it is considered negligible in nature 
(Ratledge 1978; Atlas 1981; Aeckersberg et al. 1991). Experimental results showed 



288 S. Radwan 

that the microbiological degradation of hydrocarbons in Kuwaiti Desert soil samples 
was insignificant during the long dry period of the year, but was resumed actively 
during rainy months (Radwan et al. 1995c). This result demonstrates that water is 
a major limiting factor controlling the microbiological hydrocarbon degradation in 
desert soils. Water in soil is also needed for microbial motility, microbial spacial 
proliferation and substrate transport (Smiles 1988). 

Salinity reduces water activity and thus, water availability to micro-organisms 
and other living beings. However, oil-utilizing bacteria in the desert soils and 
coastal areas seem to be adapted to the range of salinities common in their natural 
habitats. We have noticed that terrestrial oil-degrading bacteria operate optimally in 
the absence of any added sodium chloride, whereas coastal and sea water isolates 
needed about 3.5% (w/v) sodium chloride for optimal activity. 



13.9.5 Inorganic Nutrients 

Desert soils are commonly quite poor in organic matter but usually contain ade- 
quate amounts of most of the inorganic nutrients needed by micro-organisms. Under 
certain conditions, however, soil micro-organisms may show increased require- 
ments for some specific inorganic nutrients. Oil pollution is a typical example of 
such a condition. Hydrocarbons are consumed by micro-organisms as carbon and 
energy sources. In order for hydrocarbon-utilizing micro-organisms to synthesize 
proteins, nucleic acids, and other organic nitrogenous compounds, and to enhance 
their energy metabolism, they require additional amounts of nitrogen and phosphorus 
(Atlas and Bartha 1972; Gibbs 1975; Gibbs et al. 1975). It has been estimated that 
60 mg N and 6 mg P are needed for the metabolism of one gram of hydrocarbons 
(Kant et al. 1985). 

Experimental results in our laboratory showed that the microbiological hydro- 
carbon degradation in oily Kuwaiti Desert soil samples and coastal areas was 
enhanced by nitrate but not by phosphate fertilization (Radwan et al. 1995c). This 
result indicates that such soil samples contain insufficient concentrations of nitrogen, 
but adequate concentrations of phosphorus. Some authors suggested the use of 
oleophilic nitrogen and phosphorus fertilizers for enhancing oil biodegradation 
especially in the oily marine ecosystem (Atlas and Bartha 1972; Atlas 1977). 
Hydrocarbon-utilizing bacteria predominantly have the potential for using both 
inorganic and organic nitrogen sources (Radwan et al. 1995a,b, 2000). Unpublished 
results in our laboratory indicate that some of such bacteria, such as Bacillus spp., 
are capable of atmospheric nitrogen fixation, and that the symbiotic nitrogen-fixing 
nodule bacteria (Rhizobium and Brady rhizobium spp.) can utilize hydrocarbons as 
the sole sources of carbon and energy (see also Prantera et al. 2002). 

Oil-polluted desert soils and coastal areas become simultaneously enriched with 
heavy metals which are known to inhibit micro-organisms at rather low con- 
centrations (Gadd 1990). However, micro-organisms are provided with defense 
mechanisms against heavy metal toxicity. These mechanisms include the reduction 



13 Microbiology of Oil-Contaminated Desert Soils 289 

of transport of such metals across the cell envelope, their complexing and 
subsequent precipitation outside the cell, and compartmentalization inside the cell 
(Atlas and Bartha 1998). 



13.9.6 Surfactants 

Many, but not all, hydrocarbon-utilizing micro-organisms produce surfactants 
extracellularly in order to emulsify or pseudosolubilize these water-insoluble 
substrates prior to their uptake (Desai and Banat 1997; Cameotra and Makkar 1998; 
Makkar and Cameotra 1998, 2002; Banat et al. 2000). Such biosurfactants 
comprise low molecular weight compounds such as trehalose lipids, rhamnolipids, 
surfactin, polyol lipids, and fatty acids, and high molecular weight compounds such 
as emulsan, liposan, mannan, and lipoproteins. Apparently, biosurfactants and chemi- 
cal surfactants change the physical nature of oil, but do not eliminate it from the 
environment. 

There are contradicting reports on the effect of such compounds on the micro- 
biological degradation of oil substrates, as both enhancing and inhibitory effects 
were recorded (Tumeo et al. 1994; Bai et al. 1997; Lang and Wullbrandt 1999). The 
inhibitory effect was attributed to surfactant toxicity, preferential metabolism of the 
surfactants over the hydrocarbons, and/or interference with the membrane uptake 
process (Efroymson and Alexander 1991; Rouse et al. 1994; Mulligan et al. 2001). 
Several authors emphasize the need for more study before surfactant- enhanced 
bioremediation approaches may be suggested (Leavitt and Brown 1994; Van Eyk 
1994; Korda et al. 1997; Bolba et al. 1998). 



13.10 Bioremediation Strategies 

Bioremediation has been defined as the technology in which microbial activities are 
implemented to mineralize and remove xenobiotic pollutants from the environment 
(Atlas and Pramer 1990). During the 1990s a number of books and review articles 
were published on this subject (e.g., Hinchee and Olfenbuttel 1991a,b; Riser- 
Roberts 1992; Rosenberg 1993, Alexander 1994; Stoner 1994; Atlas 1995; Radwan 
et al. 1995c). Out of the xenobiotic pollutants, the hydrocarbon contaminants were 
among the first to receive close attention (Mueller et al. 1989; Song et al. 1990; 
Hinchee and Olfenbuttel 1991a,b). Bioremediation technology involves two basic 
approaches: biostimulation, or enhancing the activity of indigenous micro-organisms 
capable of degrading the pollutants, and bioaugmentation, or seeding the environment 
with pollutant-degrading micro-organisms. 

The current section of this review deals with bioremediation strategies for cleaning 
oily desert and coastal areas of the Arabian Gulf region. However, before examining 
such strategies, it may be useful to refer to earlier bioremediation attempts from 



290 S. Radwan 

other parts of the world. One such attempt was the partial bioremediation of about 
800,000 gallons of oily wastewater in the bilge tanks of the Queen Mary moored in 
Long Beach Harbor, CA (Applied Biotreatment Association 1989). There were also 
attempts to clean oily terrestrial environments in refineries and tank farms using 
bioremediation technology (Zitrides 1990). Even oil spills in the open sea were 
subjected to bioremediation attempts. In the summer of 1990, the Norwegian 
tanker Mega Borg discharged 100,000 barrels of crude oil into the Caribbean, and 
the Texan Company Alpha Environmental applied a mixture of oil-degrading 
micro-organisms in an attempt to clean up the resulting spill (McKinnon and Vine 
1991). In the spring of 1989, the Exxon Valdez tanker released about 11 million 
gallons of crude oil into Prince William Sound in Alaska, thus heavily contaminat- 
ing more than 1,000 miles of the Alaskan coasts. Because physical removal of oil 
by simple washing of the coasts was of only limited effectiveness, it was suggested 
to fertilize contaminated areas with nutrient that might enhance the potential of the 
indigenous microbial flora for biodegrading the oil (Pritchard and Costa 1991; 
Bragg et al. 1992, 1994). Reportedly, the rates of oil biodegradation were enhanced 
three to five times when contaminated coasts were fertilized with 4-8 kg nitrogen 
per 10 m 2 , and the time required for oil removal was thus reduced from 10-20 years 
to only 2-3 years. 

There are no large-scale attempts to bioremediate in situ oily desert soils and 
coastal areas in the Gulf. Yet, results from basic studies make it possible to design 
cleaning protocols. As mentioned above, bioremediation involves two major 
approaches, seeding with oil-degrading micro-organisms and fertilization with nutrients 
enhancing the indigenous microflora. As far as seeding is concerned, there are com- 
mercial mixtures of micro-organisms available in the market for in situ application 
(Applied Biotreatment Association 1989, 1990). However, experience in our laboratory 
indicates that seeding should not be the approach of choice for bioremediating oily 
environments in the Gulf (Radwan 1990; Radwan et al. 1997). Bioremediation should 
depend on indigenous strains, which are adapted to the prevailing environmental 
conditions. Oil spills create specific conditions for enhancing indigenous oil-degrading 
strains. In one of our studies (Radwan et al. 1997), we found that after a simulated oil 
spill, and for 28 weeks, desert soil samples became steadily enriched with one 
specific, indigenous, oil-degrading Arthrobacter strain, KUCC201. Other indigenous 
oil-utilizing bacteria, including other Arthrobacter strains either remained unchanged 
at low numbers or steadily disappeared. Conversely, seeding the 24-week-old 
polluted samples with local or foreign oil-degrading isolates resulted in dramatic 
decreases in the numbers of the predominant, indigenous, oil-degrading Arthrobacter 
strain, KUCC201. We concluded that seeding is probably a useless, or even harmful 
approach for bioremediation. In this context, genetically engineered oil-degrading 
micro-organisms have been created for decades (Hartmann et al. 1979; Reineke 
and Knachmuss 1979). However, the introduction of such strains into the environ- 
ment is obviously hazardous and requires governmental regulation (Halvorson 
et al. 1985). 

The above discussion indicates that the approach of choice for bioremediating 
oily environments should rely on the enhancement of activity of indigenous 



13 Microbiology of Oil-Contaminated Desert Soils 291 

hydrocarbon-utilizing micro-organisms. This may be achieved, in particular, by 
fertilization with nutrients such as nitrogen. 

As far as the oily desert soil areas are concerned, management should primarily 
include irrigation and nitrogen fertilization. The Gulf desert soils receive intermit- 
tent precipitation only during the short winter, therefore self-cleaning of oily areas 
by indigenous micro-organisms ceases most of the year (Radwan et al. 1995). Mere 
irrigation with water activates the micro-organisms in the summer, and fertilization 
with KN0 3 enhances the cleanup process considerably (Radwan et al. 1995). In 
view of these facts, a phytoremediation approach depending on rhizosphere tech- 
nology may be suggested as the biotechnology of choice for bioremediating oily 
deserts. 

Our group studied the role of rhizospheric bacteria in the attenuation of hydro- 
carbons in oily desert soils. As mentioned before, roots of desert plants and crop 
plants growing in pristine and oily soils were found to be densely colonized with 
oil-utilizing bacteria (Radwan et al. 1995a, 1998). Our group offered experimental 
evidence that cropping is a successful practice for cleaning up oily desert soils 
(Radwan et al. 2000). The crop we used was broad beans (Viciafaba), because the 
plants were able to tolerate up to 10% crude oil in soil. Common cropping practices 
such as irrigation and mechanical managements improve soil moisture content and 
aeration. Obviously, such practices provide better conditions for the growth and 
activities of soil micro-organisms. The roots of legume plants, including Viciafaba, 
carry nodules that fix molecular nitrogen, thus providing a natural and economical 
route for nitrogen fertilization. We found that the amounts of extractable hydrocar- 
bons recovered from oily desert soils supporting V. faba, were less than those 
recovered from the uncultivated soil controls. A group of rhizospheric bacteria 
known as plant growth-promoting rhizobacteria (PGPR), predominantly pseu- 
domonads, has been described to enhance plant growth when inoculated into the 
root area (Polonenko et al. 1987; Zhang et al. 1996, 1997). 

We have found that inoculation of V.faba roots with PGPR enhanced the growth 
of this plant in oily desert soil samples, and increased the phytoremediation poten- 
tial of this crop for the oily soil (Radwan et al. 2005b). In this context, we also 
found that nodule bacteria and PGPR have the potential for biodegradation of 
hydrocarbons (Radwan et al. 2007b). Thus, the rhizosphere microenvironments 
provide conditions optimal for activity of hydrocarbon-degrading bacteria. The 
rhizospheres are well aerated by the oxygen pumped down through the roots which 
may be an important factor because oxygen is a limiting factor for hydrocarbon 
biodegradation. The rhizospheres of legume crops are enriched with compound 
nitrogen fixed by the nodule bacteria, and assimilable nitrogen is also a limiting 
factor for hydrocarbon biodegradation. In addition, the rhizospheres are enriched 
with exuded vitamins and organic substances that have been recorded to enhance 
growth and activities of oil-utilizing bacteria in culture (Radwan et al. 2000; 
Radwan and Al-Muteirie 2001). 

The main technical problem involved in bioremediation of oily coastal regions is 
that any added microbial inocula or nutrient fertilizers can potentially be washed 
out into the open sea during wave and tidal movements. On the other hand, such 



292 S. Radwan 

environments do not seem to suffer much from drought. In view of these facts, we 
believe that the most promising approach for bioremediating oily coastal areas in the 
Gulf region is self-cleaning via oil-utilizing micro-organisms in biofilms (see Section 
13.6) along the coasts. Those biofilms occur in cyanobacterial mats and epilithic algal 
growth, and coat gravel particles, sand grains, and other animate and inanimate coastal 
materials. In such biofilms, micro-organisms are firmly immobilized, and thus are not 
readily washed out into the open water. Furthermore, in cyanobacterial mats and epi- 
lithic biomass, such micro-organisms are provided with oxygen and other products of 
photosynthesis such as organic nutrients including nitrogenous compounds and vita- 
mins. Our regular field trips along the oil-polluted Kuwaiti coasts after the war 
revealed that the coastal oil sediments were predominantly covered with cyanobacte- 
rial mats. Month after month, we noticed that the oil sediment layers became steadily 
thinner until they completely disappeared together with their mat covers three to five 
years after the spill. Thus, it became obvious that such natural phenomena are effi- 
cient in self-cleaning of the coasts in the Gulf region. 



13.11 Conclusions 

Desert soils and coastal areas of the Arabian Gulf contain indigenous hydrocarbon- 
utilizing micro-organisms. In desert soils, those micro-organisms are found in the 
sand and the rhizospheres of desert plants, whereas in coastal areas they occur 
mainly in association with inanimate (e.g., sand, gravel) and animate (e.g., blue- 
green mats, epilithic algal growth) materials. The predominant desert soil bacteria 
belong to the genera Micrococcus, Pseudomonas, Bacillus, Arthrobacter, and to the 
group of nocardioforms. The predominant fungi belong to the genera Aspergillus, 
Penicillium, Fusarium, and Mucor. Predominant micro-organisms in the rhizo- 
spheres are the genera Cellulomonas, Rhodococcus, and Arthrobacter. The major 
actinomycete in the desert soils is Strep tomyces. 

In general, organisms from the same genera occur in coastal areas, where they 
are mainly associated with blue-green mats in which the predominant cyanobacte- 
rial partners are the genera Microcoleus and Phormedium. Oil-pollution results in 
dramatic increases of the frequencies of these organisms. Micro-organisms in the 
desert soils suffer mainly from drought and lack of nutrients, particularly fixed 
nitrogen. Therefore, bioremediation of oily desert soils should involve activating 
indigenous micro-organisms via watering and fertilization with nitrogenous and 
easily utilizable carbon compounds. Phytoremediation, particularly by planting oily 
areas with legume crops whose roots are loaded with nodule bacteria, may be the 
approach of choice for cleaning oily desert soil. In coastal areas, which are naturally 
rich in blue-green mats, self-cleaning occurs at a satisfactory rate. 

Acknowledgements Thanks are due to Mrs. Samar Salamah and Mrs. Majeda Khanafer for their 
valuable help during the manuscript preparation. Some of the unpublished results of the author in 
this chapter have been obtained through Research Projects No.SL07/03 and RS01/04, supported 
by Kuwait University. 



13 Microbiology of Oil-Contaminated Desert Soils 293 

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Chapter 14 

Microbial Communities in Fire-Affected Soils 

Christopher Janzen and Tammy Tobin-Janzen(S) 



14.1 Introduction 

Of all the ways in which human activities can affect soil environments, fires are per- 
haps the most dramatic. Whether set deliberately or accidentally, the worldwide 
impact that such fires have is extensive, including the loss of human and animal lives 
as well as economic and ecological damage (UNECE et al. 2000; UNECE and FAO 
2001; Davidenko and Eritsov 2003; Kudoh 2005; FAO 2005). The United Nations 
Food and Agricultural Organization (2005) estimated that 350 million hectares burn 
annually, and that approximately 90% of those fires are of human origin. The 
frequency and severity of surface fires have also increased in many parts of the world 
due to changes in climate and land management practices (Houghton et al. 1992; 
Renkin and Despain 1992; Glantz 1996; Neary et al. 1999; Westerling et al. 2006). 
Large fires tend to draw media attention, particularly when they impinge on densely 
inhabited or well-known wildlife areas. Their rapid spread, extreme temperatures, and 
the barren landscapes they leave behind can alter the surrounding ecosystem for 
years, decades, or even permanently. It is no surprise, therefore, that a premium has 
historically been placed on extinguishing fires as they happen, rather than on studying 
their ecological significance. Only recently have scientists begun to understand the 
critical roles that some fires play in sustaining natural environments, and on the parts 
that soil micro-organisms play in that process. 

Two basic types of fires are discussed in this chapter: surface fires and 
underground fires. Surface fires include both wildfires and prescribed fires and their 
effects are predominantly 'top down'. That is, the fire source is aboveground, and 
the heat from the fire, although it can be intense enough to sterilize the surface soil, 
may not penetrate more than a few centimeters below the soil surface. Surface fire 
effects often consist of a patchwork of severely affected sites interspersed with less 
affected areas, the pattern of which is dictated by the availability of fuel, topography, 
and other factors. 



Tammy Tobin-Janzen 

Biology Department, Susquehanna University, Selinsgrove, PA17870, USA 

e-mail: tobinjan@susqu.edu 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 299 

© Springer- Verlag Berlin Heidelberg 2008 



300 C. Janzen and T. Tobin-Janzen 

Underground fires, by way of contrast, have a 'bottom-up' impact. They are 
typified by the large coalmine fires found in the Jharia Coalfield of India (Agarwal 
et al. 2006), throughout much of northern China (Zhou et al. 2006), and throughout 
the coal-mining regions of the United States, most notably in Centralia, Pennsylvania 
(Trifonoff 2000). In these fires, steam and gases carrying vaporized combustion 
products from the burning coal below migrate, or 'vent', upward through soil 
fractures, cooling as they rise. As the gases from these 'anthracite smokers' cool, 
their dissolved chemicals either escape to the atmosphere or condense into the 
surrounding soils. Thus, underground fires are distinguished by subsurface soil 
temperatures that are generally much hotter than surface temperatures, and by soil 
chemical changes that tend to be clustered around actively venting soil fractures. 
In many ways, these fires resemble geothermal environments as closely as they do 
environments affected by surface fires. In this chapter, we use coalmine fires as the 
model system for underground fires in general. 

Regardless of whether a fire has a top-down or a bottom-up impact, it alters 
many surface and subsurface soil properties as it migrates through an area. These 
properties include the availability of water, the structure and composition of soil 
particles, and the availability of nutrients. Sulfur, nitrogen, phosphorus, and carbon 
species, in particular, can experience dramatic shifts when pre- and post-fire envi- 
ronments are compared. 

The extent and severity of these fire-induced changes is determined by many 
factors including the duration of the fire, the intensity of the fire, the rate of the 
fire's spread, the topography of the burn area, the initial soil composition, the soil 
moisture content, fuel characteristics, weather conditions, the interval between fire 
events, and the frequency of fire events. However, the greatest of these is duration. 
A fast-moving aboveground fire of even moderate intensity does not persist long 
enough to transfer enough heat to the soils to cause substantial belowground 
chemical changes. Slow-moving fires cause severe and complex changes that can 
be long lasting or permanent. Fire temperatures can range from 50 to >1,500°C. 
Heat release can range from 2.11kJ/kg of fuel to 2.1MJ/kg. The effect of fire 
intensity is related to how well the energy produced by the fire is transferred to 
the soil. A slow-moving intense fire will have a greater impact than a fast-moving 
fire of the same intensity. Likewise, a slow-moving fire of low intensity may have 
as great an impact as a fast-moving fire due to the greater length of time the fire 
will be transferring the generated heat to the soil. 

Ultimately, it is both the intensity and persistence of fires, as well as the resulting 
changes to the surface and subsurface soil properties that determine the fire's 
impact on resident microbial communities. Although these communities are 
likely to play critical roles in both post-fire biogeochemical nutrient cycling and 
in the ultimate recovery of fire- affected environments, they have remained quite 
poorly studied. Most prokaryotic research, in particular, has focused on studies of 
post-fire microbial biomass, microbial metabolic assays, and culture-dependent 
assays (Ahlgren 1974; Dunn et al. 1979; Bissett and Parkinson 1980; Klopatek 
and Klopatek 1987; Klopatek et al. 1990; Fritze et al. 1993; Vazquez et al. 1993; 
Acea and Carballas 1996; Hernandez et al. 1997; Ross et al. 1997; Prieto-Fernandez 



14 Microbial Communities in Fire-Affected Soils 301 

et al. 1998; Choromanska and DeLuca 2002; Andersson et al. 2004; Treseder et al. 
2004; Naumova 2005; Giai and Boerner 2007). Only recently have culture- 
independent methods such as 16S rRNA gene sequencing, terminal restriction 
fragment length polymorphism (T-RFLP) analysis, phospholipids fatty acid 
analysis (PLFA), and denaturing gradient gel electrophoresis (DGGE) been 
combined with more traditional methods to give a more complete picture of the 
effects that fires have on specific prokaryotic communities, and on the roles 
that those communities play in the post- fire environment (Baath et al. 1995; 
Pietikainen et al. 2000; Jaatinen et al. 2004; Tobin-Janzen et al. 2005; Yeager 
et al. 2005; Izzo et al. 2006). 



14.2 The Effects of Surface Fires on Microbial Environments 

Because the fire-induced thermal, chemical, and physical changes to the soil 
environment directly drive microbial community responses, it is important to 
understand these factors before turning to a more complete discussion of microbial 
ecology. The soil changes that most clearly affect microbial community responses 
depend on the intensity of the fire, and correspond to changes in the availability of 
water, nitrogen, sulfur, phosphorus, and carbon. 



14.2.1 Fire Intensity 

The intensity of a surface fire, and its resulting impact on the microbial communities 
in underlying soils, varies according to the total amount of heat produced by the fire, 
the mode of transfer of that heat to the underlying soils, and the speed with which the 
fire progresses through an area. The total heat produced by the fire is governed by the 
nature of the fuel source and the fuel load. For example, grassland fires (Raison 1979; 
Ross et al. 1997; Neary et al. 1999) tend to have lower fuel loads and thus generate 
lower fire temperatures than fires in tropical woodlands or conifer forests. Because 
microbial mortality generally begins at around 50°C, but varies tremendously from 
species to species, the total heat produced by the fire is of paramount importance in 
determining the post-fire microbial community composition. 

The modes of energy transfer from a surface fire to the soil are radiation, 
convection, conduction, vaporization/condensation, and mass transport (Neary et al. 
1999). Electromagnetic radiation from the fire can play a significant role in energy 
transfer at the onset of a burn. Movement of air masses by convection can transfer 
heat from the active fire to nearby areas, heating nearby soils as well as spreading 
the fire. Conduction of heat by direct contact between hot or burning fuel and 
cooler soil can be important especially where fuel loads are heavy or where fuels 
themselves are massive such as with slash piles. The high specific heat capacity of 



302 C. Janzen and T. Tobin-Janzen 

water (4. 184 J g~ l0 C _1 ) means that a substantial amount of energy is required to 
vaporize the water in soil. The enthalpies of heating and vaporization of soil moisture 
tend to mitigate any rise in soil temperature until all the water is vaporized. Indeed, 
the soil temperature cannot rise above 95 °C until vaporization is complete 
(Campbell et al. 1994). The vaporized water carries latent heat through soils faster 
and deeper than other modes of energy transport such as radiation or conduction. 
Thus, whereas moist soils initially suffer lower temperatures, the overall impact of 
the fire may be more rapid and affect soil to a greater depth. The transfer of energy 
via mass transport generally has little to no effect on soil temperatures. 

Once all of the moisture in soils under a fire has vaporized, soil temperatures 
typically rise to 200-300°C. However, under severe or slow-moving fires, surface 
soil temperatures can rise to as high as 700°C (DeBano et al. 1998). The depth- 
temperature profile for a soil under a fire is determined by intensity and duration 
of the fire as well as the moisture content of the soil. With fast-moving or low 
severity fires, belowground soil temperatures generally do not exceed 100-150°C 
at 5cm depth and demonstrate no heating below 30cm (Agee 1973; DeBano 
2000). A slow-moving or intense fire will clearly have a more significant effect on 
both the maximum soil temperatures observed and the depth at which tempera- 
tures are raised. 



14.2.2 Available Moisture 

Available soil moisture levels are not only decreased initially as a direct result of 
vaporization, but may also suffer long-term reductions. At moderate temperatures, 
incomplete burning of organic matter can result in the formation of a hydrophobic 
coating in the mineral components of soil resulting in increased repellency and 
decreased soil permeability. This repellency can result in a persistent decrease in 
soil moisture in the post-burn soils or in greater run-off and erosion. Despite these 
tendencies, situations in which soil moisture has increased (Klock and Hevey 1976; 
Haase 1986) or remained the same (Campbell et al. 1977; Milne 1979) after fire 
have also been documented, and thus the overall impact that post-fire soil moisture 
has on microbial communities is varied (Letey 2001). 



14.2.3 Available Nutrients 

In an aboveground fire, the most dramatic and well-understood changes to available 
nutrients involve the volatilization, chemical transformation, and biogeochemical 
cycling of nitrogen, carbon, phosphorus, and sulfur. Although changes can and do 
occur to trace minerals such as As, Ca, K, Na, Fe, and Al (Grier 1975; Feller 1982; 
Macadam 1989; DeBano et al. 1998; Neary et al. 1999; Arocena and Opio 2003) 
the effects that these changes have on microbial communities remain mostly 
unstudied, and thus are not discussed in this chapter. 



14 Microbial Communities in Fire-Affected Soils 303 

14.2.3.1 Nitrogen 

At moderate temperatures, dead partially combusted plant and microbial biomass can 
be easily oxidized resulting in an increase in inorganic nitrogen immediately post burn 
(Diaz-Ravina et al. 1996). More extreme temperatures can result in volatilization of 
nitrogen (Giovannini et al. 1990). Total nitrogen decreases slightly as the temperature 
rises from 25 to 220°C but dramatically decreases between 220 and 460°C (Giovannini 
et al. 1990). Ammonium concentrations steadily increase up to 220°C but, like total 
nitrogen, drop quickly as the temperature rises above 220°C until very little remains at 
460°C. It is presumed that this increase in NH+-N is due to mineralization of organic 
nitrogen. Nitrate concentrations are initially unaffected by fire, even moderate to 
intense fires. However, N0 3 "-N is produced by biochemical nitrification of ammonium 
in the time following the fire resulting in N0 3 "-N concentrations that can be 
significantly higher in the weeks or years post-fire (Covington et al. 1991). 

Therefore, although total nitrogen may decrease, the bioavailable forms of nitro- 
gen, nitrate and ammonium, may be at elevated levels for several years following a 
burn. Ammonium tends to adsorb onto the mineral soil and become immobilized. 
Unless regrowth of vegetation occurs soon after the fire, the nitrate can be easily 
lost to leaching, thus depleting the total nitrogen for long periods. Conversely, if 
regrowth is rapid, soil organic nitrogen concentrations can rapidly recover to 
pre-fire levels (Adams and Attiwill 1984; Weston and Attiwill 1996). 

14.2.3.2 Carbon 

Fire severity as described by the maximum sustained surface temperature under a fire 
affects the degree of consumption of litter and soil organic matter. A low severity burn 
where the soil temperatures do not exceed 250°C can cause partial scorching of litter. 
As depth in the soil increases, the insulating ability of the soil mitigates the heating 
of soils. Thus, with surface temperatures at or below 250°C, soil temperatures down 
to 2.5 cm rarely exceed 100°C and at 5 cm the temperature is typically below 50°C. 
The lower temperatures at depth result in only partial distillation of organic matter 
above 2.5 cm and little to no effect below 5 cm. A moderate burn characterized by 
surface temperatures up to 400°C results in temperatures at 2.5 and 5 cm of up to 175 
and 50°C, respectively. These temperatures result in very significant charring of litter, 
some charring of organic matter to 2.5 cm and the start of distillation of organic matter 
above 5 cm. In a severe burn, surface temperatures exceed 675°C, resulting in 
complete combustion of the litter. The soil down to 2.5 cm can experience heating to 
190°C with concomitant charring or consumption of large portions of the organic 
matter. Even at depths of 5 cm, soils are being heated to 75°C resulting in significant 
distillation of volatile organic matter and some charring (DeBano et al. 1977). 

14.2.3.3 Phosphorus 

Organic phosphorus is readily converted to inorganic forms of phosphate. Even in 
low severity fires where temperatures do not exceed 200°C, the concentration of 



304 C. Janzen and T. Tobin-Janzen 

organic phosphorus decreases markedly. In an artificial heating experiment, organic 
phosphorus was completely depleted from soils above 220°C (Giovannini et al. 
1990). Organic carbon is easily lost, however, total phosphorus remains fairly con- 
stant due to its low volatility. The organic phosphate is instead converted to 
orthophosphate, the predominant form of bioavailable phosphorus (Cade-Menun 
et al. 2000). The concentrations of inorganic and available phosphorus peak at soil 
temperatures of approximately 450°C (Giovannini et al. 1990). 

Other factors play a role in determining how much of the inorganic phosphorus 
produced by a burn is available. Bioavailability peaks at a pH of around 6.5 (Sharpley 
2000). The ash produced in a fire tends to move the soil pH higher toward this value. 
However, in calcarerous soils phosphate can complex very strongly to calcium, 
resulting in removal of phosphorus from the available pool. In acidic soils, phosphate 
can bind with other soil metals such as iron, manganese, and aluminum. In summation, 
aboveground fires easily convert most or all of the organic phosphorus to inorganic 
phosphorus, thus initially increasing the bioavailability. How long the increase in 
bioavailable phosphorus persists depends on soil pH, the composition of the mineral 
soil, and the rate of uptake of available phosphorus by recolonizing vegetation. 

14.2.3.4 Sulfur 

In an artificial heating study, Badia and Marti (2003) found that a slight steady 
increase in total sulfur occurred upon heating gypsiferous soils up to 500°C and 
upon incorporation of ash into the soil, but that no change in total sulfur occurred 
in calcarerous soils. In another study, Castelli and Lazzari (2002) found that total 
sulfur peaked a year after a controlled burn in both grass-covered and shrub- 
covered soils. After a second controlled burn three years after the first burn, the 
total sulfur in the soils under grass cover dropped back to the original (before 
the first burn) levels below 1 cm depths. In the same study, available sulfur under 
shrub cover immediately jumped significantly, but then dropped back to pre-burn 
levels within two years. The available sulfur under grass cover was not affected. In 
both cases, the available sulfur did not change with the second controlled burn. 
These two studies indicate that the sulfur chemistry of soils is affected by above- 
ground fires in manners similar to other nutrients. 



14.3 The Effects of Underground Fires on Microbial 
Environments 

Underground coalmine fires have very different effects on soil chemistry than those 
caused by aboveground fires. The reasons stem from three major factors. The heat 
source is below the surface, the duration of the belowground fires can be consider- 
ably longer than surface fires, and the composition of the coal fuel is quite different 
from that found in typical prescribed or wild aboveground fires. These factors lead 
to unique physical and chemical properties and processes. 



14 Microbial Communities in Fire-Affected Soils 305 

14.3.1 Fire Intensity 

In an aboveground fire, the highest temperatures the soils experience are at the 
air-surface interface. Conversely, the lowest temperature of an affected soil is at 
the air-surface interface with a belowground fire. In other words, the soil 
temperature gradient in a belowground fire is inverted from that observed in 
aboveground fires. 

In addition, the impact of the fire is not evenly distributed spatially. The 
primary modes of energy transfer from the fire to the surface and near- surface 
soils are convection and conduction. Convection of hot combustion gases takes 
the paths of least resistance, following the small fractures and faults in the 
subsurface. The gases tend to escape to the atmosphere in vents. The temperature 
of the surrounding soils is highest near the vent as a result of the transfer of 
thermal energy from the hot gases to the surrounding soils. These hot soils are 
then able to transfer heat away from the vent via conduction. Eventually, this heat 
is slowly lost to the atmosphere. Thus, the surface temperature decreases with 
increasing distance from the vent. In addition to heat brought to the surface by 
convection, the hot subsurface rock and mineral matter can transfer heat to the 
surface directly by conduction. This conduction results in surface regions that 
are quite hot while displaying no outward signs of venting of hot gases. Indeed, 
the authors have measured surface temperatures above the Centralia Pennsylvania 
mine fire exceeding 400°C in nonventing areas (Tobin-Janzen, unpublished 
results). The underground environment (including both structure and composi- 
tion) is not uniform. Therefore, the heat transferred by conduction is spatially 
uneven. The end result of the modes of heat transfer is that the surface tempera- 
tures above an underground fire are distributed in an irregular pattern. 

Another significant difference between aboveground and belowground fires is 
the duration. Aboveground fires persist at a certain location for hours to weeks. The 
available fuel is consumed quickly and when gone, no further burning is possible. 
Stated another way, aboveground fires are fuel limited. The rate of progression of 
belowground fires is much slower, and can range from ten meters per year to 
several hundred meters per year. Even at the fastest rates, underground fires 
progress more slowly than aboveground fires. This slow movement means that an 
area on the surface that becomes affected by the underground fire may experience 
these effects for years. Indeed, certain areas in Centralia have been affected contin- 
uously for decades (Trifonoff 2000). 

The principal reason for the slow movement is a shortage of oxygen. That is, 
belowground fires are oxidant limited. Oxygen is replenished through the fractures 
in the subsurface structures. Indeed, weather systems affect the progress of the fire. 
Low-pressure weather systems result in more venting of gases whereas little 
venting takes place during high-pressure systems. Another consequence of this 
shortage of oxygen is that the underground fire is a reducing fire. Thus, the reduced 
and volatile forms of nitrogen (ammonium) and sulfur (S° and H 2 S) are formed and 
transported to the surface. 



306 C. Janzen and T. Tobin-Janzen 

14.3.2 Available Moisture 

In general, as the hot combustion products of the fire rise through the mineral and 
soil column, those components with low volatilities begin to condense. One simple 
manifestation of this phenomenon is the formation of a very moist semi-liquid layer 
beneath active vents. When the hot gas encounters cooler soil, water condenses 
creating a very hot mud between 0.5 and 1 m beneath the surface. (Tobin-Janzen, 
unpublished results). Above this level, the soil moisture varies tremendously, and 
the soil can even be quite dry, more closely resembling soils affected by above- 
ground heat sources (Tobin-Janzen et al. 2005). 



14.3.3 Available Nutrients 

The chemical composition of coal is markedly different from the organic fuel 
burned in aboveground fires. Coal is a highly variable fossilized form of ancient 
plant matter that has been significantly altered by exposure to elevated temperature 
and pressure. In addition to the organic components, coal contains significant 
inorganic materials. When burned, the inorganic components can melt and volatilize. 
Indeed, these inorganic phases pose technological problems for the use of coal as a 
clean energy source. The combustion products, including the inorganic phases, can 
percolate to the surface and near- surface environments, where they have been 
shown to condense into a variety of minerals, including elemental sulfur, downeyite 
(Se0 2 ), orpiment (As 2 S 3 ), laphamite (As 2 (Se,S) 3 ), ammonium chloride, gypsum 
(CaS0 4 H 2 0), and mullite (Al 6 Si 2 13 ; Finkelman and Mrose 1977; Lapham et al. 
1980; Dunn et al. 1986). Indeed, the authors have employed X-ray powder 
diffraction to identify pure ammonium chloride and needlelike elemental sulfur 
crystallizing on the surface of the ground above the Centralia Pennsylvania mine fire 
(Janzen, unpublished results). Although the rate of deposition of these compounds 
is not high, the impact caused by a long and slow-burning underground coalmine 
fire has the potential to be significant. Also, as described above, the distribution of 
combustion gases and volatile chemical species at the surface is not even. Rather, 
it is a function of the subsurface structure. Thus localized areas of high chemical 
concentrations, temperature, and soil moisture are interspersed with areas of 
lower concentrations and more moderate temperatures. 



14.3.3.1 Nitrogen 

Tobin-Janzen et al. (2005) studied the inorganic nitrogen (NH+-N and N0 3 "-N) 
levels in surface soils overlying the Centralia, Pennsylvania coalmine fire over a 
two-year period. During that time, the mine fire front progressed through the 
sample site, and surface soil temperatures increased an average of 16.1 °C. Many of 
the hottest areas showed elevated ammonium (as high as 13.79mg/kg) and nitrate 



14 Microbial Communities in Fire-Affected Soils 



307 



(as high as 103.1 mg/kg) levels, however, these concentrations were not correlated 
with absolute temperature values, nor to proximity to an active vent, as some areas with 
elevated temperature, close proximity to an active vent, or both, also had inorganic 
nitrogen concentrations that were comparable to those in nearby unaffected soils. 
Rather, inorganic nitrogen levels were related to more complex environmental 
trends, such as the length of time an area had been affected by the mine fire and 
whether the overall soil temperatures were rising or falling in the area due to the 
movement of the mine fire front. 



14.3.3.2 Sulfur 

The sulfur-containing gases percolating up from the burning coal have a profound 
effect on the soil chemistry of the overlying soils, and thus represent one of the most 
striking differences between underground mine fire-affected soils and those affected 
by surface fires. For example, in unpublished work, the total sulfur concentrations 
are clearly affected by the underground fire (Fig. 14.1). The concentrations are sig- 
nificantly higher near the vent. Interestingly, the sulfur concentrations are not high- 
est directly at the vent. Rather, they increase to a maximum approximately 0.5 m 



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'.S'13»i 



] 



-1.0 -OS -02 02 

Distance from Center of Venl (m) 



Fig. 14.1 Spatial distribution of total sulfur around an active vent above the Centralia, 
Pennsylvania mine fire. The concentrations of total sulfur were measured in dry soil on two days 
during the summer of 2005. The results are correlated with soil temperature at a depth of 5 cm and 
distance from the vent. Triplicate analyses were performed on each sample and the error bars 
represent the 95% confidence intervals 



308 C. Janzen and T. Tobin-Janzen 

from the vent, then decrease with decreasing distance from the vent. At greater dis- 
tances from the vent the concentrations of total sulfur again begin to drop due to the 
depletion of volatile sulfur components of the gases at greater distances from the 
source. Similar results have been observed for elemental sulfur (S°). 

Using thermodynamic principles, Stracher has proposed a stability relationship 
between the partial pressure of S 2 gas and temperature (Stracher 1995). He 
developed a model predicting which phase, orthorhombic or S 2 gas, would be stable 
under a given set of conditions. Depending on the partial pressure of S 2 , the 
temperature at which the solid form of elemental sulfur becomes the favored 
species varies. At very low partial pressures of S 2 , sulfur prefers to be solid at 
low temperatures. As the pressure rises, the solid form becomes favored at higher 
temperatures. Thus, at the vent where temperatures are highest, the gaseous form is 
predominant. Moving a short distance from the vent, the temperature is lower, 
allowing sulfur to solidify. Farther from the vent, the sulfur has already been 
depleted in the effluent gas, thus no further deposition is possible. 



14.4 Microbial Communities in Fire- Affected Soils 

The response of microbial communities to fire varies primarily with the nature of the 
fire. The fire may have a top-down or a bottom-up profile, its intensity and duration 
may vary, as also vary the chemical, physical, and biological changes it generates in 
the surrounding environment. Not surprisingly, the fire's intensity and persistence 
tend to be the most important factors in governing post-fire microbial community 
numbers and diversity. Nevertheless, other factors, such as the release of organic 
toxins, including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans 
(PCDFs), and poly cyclic (or poly nuclear) aromatic hydrocarbons (PAHs), into the 
surrounding soils can dramatically and negatively affect microbial biomass and 
diversity (Kim et al. 2003). Conversely, the increase of inorganic nitrogen and sulfur 
levels in fire-affected soils can allow microbes capable of exploiting these molecules 
to thrive in many post-fire environments, particularly if they are able to withstand the 
fire's high temperatures or to recolonize fire-affected areas quickly as they cool. 



14.4.1 Fire Intensity and Frequency 

The fire's intensity, as determined by its total heat, the transfer of that heat to the 
under- or overlying soils, and the migration rate of the fire, is generally the most 
important single factor driving microbial community responses in most environ- 
ments (Neary et al. 1999; Hart et al. 2005; Tobin-Janzen et al. 2005). Heat from 
the fire is by itself often high enough to sterilize soils, with surface temperatures 
above mine fires exceeding 730°C (Lapham et al. 1980), and those in surface fires 
capable of exceeding 675 °C. Even nonsterilizing heat levels can directly lyse bacterial 



14 Microbial Communities in Fire-Affected Soils 309 

cells, and reduce their reproductive capabilities (Covington and DeBano 1990). As 
a result, total microbial numbers as well as microbial community diversity tend to 
decrease dramatically immediately post- fire (Dunn et al. 1979; Fritze et al. 1993; 
Prieto-Fernandez et al. 1998; Tobin-Janzen et al. 2005), although some exceptions 
to this rule have been observed (Newman et al. 2003). 

Soil moisture, which varies with both above- and belowground fire conditions as 
previously described, can intensify the fire's impact on microbial cells. Moist heat 
is much more efficient at killing soil micro-organisms than dry heat, with threshold 
values for fungal and bacterial survival estimated at 80°C and 60°C for fungi, and 
120°C and 100°C for bacteria in dry and moist soils, respectively (Dunn and 
DeBano 1977; Dunn et al. 1985). Thus, increased soil moisture can be responsible 
for augmenting a fire's short-term impacts on microbial communities. Conversely, 
decreased soil moisture can exacerbate a fire's long-term impacts. Summer 
droughts, for example, have been proposed to be responsible for the slow recovery 
of bacterial and fungal community biomass following both surface and underground 
fires (Cilliers et al. 2005; Tobin-Janzen et al. 2005; Yeager et al. 2005). Finally, the 
condensing steam from underground mine fires moistens the overlying soils, and 
may actually help to increase microbial survival in fire- affected soils, even during 
summer droughts (Tobin-Janzen et al. 2005). 

The migration rate of a fire likewise determines its impact on soil microbial 
communities. Low intensity, rapidly moving surface fires do not generally exceed 
surface temperatures of 250°C, and microbes at depths as shallow as 25 mm 
generally survive. By contrast, in high-intensity surface fires with surface tempera- 
tures exceeding 675 °C, microbes as deep as 50 mm underground experience 
selective die-off (Neary et al. 1999). By comparison to surface fires, underground 
fires tend to move very slowly, and their effects can last for decades or longer. The 
Centralia mine fire, which started in 1962, is expected to burn for at least another 
100 years, and the Jharia coalmine fire has been burning in India since 1916. 

Thus, the long-term effects on soil microbial communities in these areas, 
although yet unstudied, are expected to be extensive. Over a one-year period, our 
laboratory (Tobin-Janzen et al. 2005) used T-RFLP analysis of domain Bacteria 
16S rRNA to ascertain that the complexity of microbial communities decreased 
uniformly as temperatures in the site increased. Furthermore, these changes were 
not correlated with other environmental factors such as soil pH, soil moisture, 
inorganic nitrogen, or total sulfur, but rather with general trends in the mine fire 
progression itself. It will be of interest to determine what happens to these micro- 
bial communities as the impact time stretches from months to decades. 

Another important manner in which the intensity of surface fires and underground 
fires differ from each other is in the bottom-up nature of underground fires. Because 
the fire source is belowground, subsurface temperatures become progressively 
more elevated as soil depth increases. Although the subsurface microbial communi- 
ties below 50 cm have not yet been extensively assayed in our laboratory, our 
unpublished observations suggest that as temperatures increase with depth, the total 
microbial biomass, as determined by the amount of bacterial DNA that can be 
extracted from the soil samples, decreases as well. Despite this decrease, we have 



310 C. Janzen and T. Tobin-Janzen 

successfully amplified domain Bacteria 16S rRNA genes from soil samples at 
depths of up to 50cm, and at temperatures of up to 87°C. 

The overall impact of a fire's intensity on microbial communities varies tremen- 
dously with the species composition of that community. Pietikainen et al. (2000) 
demonstrated that bacteria tend to be more resistant to fire-induced heat than fungi. 
However, not all bacteria are similarly resistant, nor are all fungi similarly susceptible. 
In fact, Izzo et al. (2006) used greenhouse experiments to demonstrate that certain 
species of ectomycorrhizal fungi may actually compete more successfully for roots 
in the presence of fire, and Neurospora ascospores germinate in response to heat or 
fire (Emerson 1948; Jesenska et al. 1993; Pandit and Maheshwari 1996). 

Heterotrophic bacteria often show the sharpest declines in numbers following 
fires, presumably as a result of the combustion of readily available food sources. 
By contrast, autotrophic bacterial numbers can actually increase as nutrients are 
released from their organic forms. In our work above the Centralia coalmine fire, for 
example, autotrophs are frequently dominant members of the bacterial communities 
present in fire-affected soils. However, Nitrobacter are generally more heat- sensitive 
than heterotrophic bacteria, (Dunn and DeBano 1977; Dunn et al. 1985), and 
actinomycetes are generally more heat-resilient than other culturable heterotrophs 
(Cilliers et al. 2005). Recent experiments have shown that many of the most common 
bacteria in fire-affected soils include endospore formers (Ahlgren 1974; Moseby 
et al. 2000; Yeager et al. 2005). This finding is not surprising, as these endospore 
formers probably survive short-term intense heat as endospores, and then rapidly 
germinate once soil moisture, temperature, and nutrient levels have returned to 
sustaining levels. Ultimately, thermophilic bacteria are the most resilient of all micro- 
bial species, and are often preferentially selected by fire conditions. Our team and 
others have demonstrated that they often make up the dominant populations in fire- 
affected soils (Norris et al. 2002; Tobin-Janzen et al. 2005; Yeager et al. 2005). 



14.4.2 Nitrogen-Cycling Bacteria 

Available nitrogen is often a limiting nutrient in pre-fire environments (Allen et al. 
2002; Yeager et al. 2005). However, both surface and underground fires can release 
inorganic nitrogen as a result of the combustion of organic molecules. This nitrogen 
initially exists as NH+-N, but is quickly oxidized to NO~-N (DeLuca and Zouhar 
2000; Tobin-Janzen et al. 2005; Yeager et al. 2005). Soil bacteria are probably critical 
components in this oxidation, and yet they have remained poorly studied in post- 
fire environments until recently. Yaeger et al. (2005) used molecular analysis of 
nifil and amoA genes to study nitrogen-fixing and nitrifying bacteria following a 
forest fire. They demonstrated that although there was a decrease in overall micro- 
bial biomass, including that of nitrogen-cycling bacteria, the nitrogen-fixing 
community actually became more diverse within a month after the fire. By contrast, 
a single ammonia-oxidizing type, belonging to Nitrosospira spp. cluster 3 A, 
dominated in the post- fire soils. 



14 Microbial Communities in Fire-Affected Soils 311 

Our laboratory has similarly demonstrated the presence of ammonia-oxidizing 
bacteria, as determined by the presence of amoA genes, in hot mine-fire affected 
soils with elevated NH+-N and N0 3 ~-N, and at temperatures of up to 60°C (Tobin- 
Janzen et al. 2005). Furthermore, we have isolated spore-forming Geobacillus that 
can reduce nitrate to N 2 at 60°C (Kauffman and Tobin-Janzen 2005). Thus, nitrogen- 
cycling bacteria appear to be important components of both surface and under- 
ground mine fire environments, where they are most likely exploiting the elevated 
nitrogen levels, and where they could be contributing to greenhouse emissions in 
the fire areas. 



14.4.3 Sulfur-Cycling Bacteria 

Perhaps the most interesting difference between environments affected by 
belowground versus surface fires is the high levels of sulfur that can condense into 
the soils above the former. In this regard, subsurface fires tend to more closely 
resemble fumaroles and other similar geothermal environments, in which surface 
soils are affected by hot gases containing varying levels of H 2 0, C0 2 , S0 2 , H 2 S, H 2 , 
N 2 , CO, and CH 4 (for further details, see Delmelle et al. 2000; Giggenbach and 
Sheppard 1989). 

Janzen et al. (unpublished results) have recently studied the sulfur levels sur- 
rounding an active vent above the Centralia coalmine fire, where they demonstrated 
that the highest levels of sulfur did not necessarily correlate with the highest soil 
temperatures, but rather with temperatures that favored condensation of the sulfur 
into the soils, rather than its loss to vaporization (Fig. 14.1). In order to determine 
if sulfur-metabolizing bacteria were capitalizing on the high levels of sulfur in these 
soils, our laboratory collected soil samples from the same boreholes, and identified 
total bacterial community members using 16S rRNA gene sequencing. 

In Fig. 14.2, it can be seen that the bacterial community members of this fire- 
affected soil are in fact enriched for thermophilic bacteria, including thermophilic 
sulfur metabolizers, nitrogen-cycling bacteria, and endo spore-forming bacteria. 
The community itself was only modestly diverse, with the dominant bacterial 
groups including Actinobacteria, Alphaproteobacteria, Deltaproteobacteria, 
Betaproteobacteria, along with Acidothermus, Clostridium, and Geobacillus 
species. These results are in good agreement with the trends seen in other hot, fire 
and geothermally affected soil environments (Norris et al. 2002; Yeager 
et al. 2005). 



14.5 Conclusions 

Although the micro-organisms capable of surviving and thriving in fire-affected 
areas are poorly studied at this time, studying their life cycles and environmental 
effects is likely to produce important discoveries. Microbes found in fire-affected 



312 



C. Janzen and T. Tobin-Janzen 




Thermaerobacter (T) 

Clone U5 

Clone C2 

uncultured bacterium (Fe) 

Clone Ut 

uncultured Acidobacreria bacterium 

uncultured Holophaga sp. 

Rhodothermus marinus(T) 



uncultured bacterium 

uncultured Addobacteria bacterium 

uncultured sludge bacterium (N) 

candidate division OPB (T) 

Clone 12 

uncultured deMa proteo bacterium 

uncultured Nitrospfra(N) 

uncultured Hoiophaga 
ncultured forest soil bacterium 

Clone A2 

Clone C1 

uncultured thermal soil bacterium (T) 

!M| Fen-ibacter thermoautclrophicus {T, Fe) 

I ' Thermolithobacter carboxydivorans {T, Fe) 

I uncultured rumen bacterium 

uncultured Thermovenabulum (N, S T Fe) 

Ammonia Oxidizing Isolate (T, N T E) 

Tnermomicrobium roseum (T) 

Sphaerobacter thermophilics (T) 

Clone At 

Clone A3 

Therm ua ap^T, S f Fe) 



Fig. 14.2 Dendrogram of 16S rRNA gene sequences obtained from the field samples sur- 
rounding the active vent depicted in Fig. 14.1. Samples designated 'Clone C and 'Clone I' origi- 
nated from the boreholes one meter to the left and right of the active vent, respectively. Samples 
designated Clone A and Clone U came from an affected surface site (50°C) located outside of 
the study area depicted in Fig. 14.1. Taxa containing known thermophiles (T), endospore-form- 
ers (E), and nitrogen (N), sulfur (S), and iron (Fe) cycling bacteria are indicated. The dendro- 
gram was generated using nearest neighbor analysis and the CLC Combined Workbench 
Program (CLC Bio). Bootstrap values from 100 resamplings are shown above each internal 
node. Thermaerobacter 16S rRNA was used as the outgroup 



areas most likely play critical roles in the biogeochemical cycling of nitrogen and 
in rhizosphere ecology, and thus can be expected to play important roles in the 
recovery of fire-affected ecosystems. Thermophilic bacteria, in general, have 
already provided biotechnology with some of its most powerful tools, and ther- 
mophilic sulfur-metabolizing bacteria are currently at the heart of several studies 
geared toward producing more efficient methods of bioleaching metals and desul- 
furizing flue gases (Huber and Stetter 1998; Kaksonen et al. 2006). Inasmuch as 
these bacteria appear to be enriched in hot mine-fire affected soils, continued 
research into their biology, environmental roles, and metabolism will be particu- 
larly interesting. Finally, not all fire-associated bacteria are benign. Iron and sulfur- 
metabolizing bacteria catalyze the rate-limiting step responsible for acidification of 
drainage effluents in coal mining environments, and the nitrogen-cycling bacteria 
identified in this study and others may also be responsible for releasing the green- 
house gases NO and N 2 into the environment. Thus, further study of these inter- 
esting extremophiles holds the promise of beneficial technological advances in a 
variety of compelling areas. 



14 Microbial Communities in Fire-Affected Soils 313 

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Zhou F, Ren W, Wang D, Song T, Li X, Zhang Y (2006) Application of three-phase foam to fight 
an extraordinarily serious coal mine fire. Int J Coal Geo 67:95-100 



Chapter 15 

Endophytes and Rhizosphere Bacteria of Plants 

Growing in Heavy Metal-Containing Soils 

Angela Sessitsch(S) and Markus Puschenreiter 



15.1 Introduction: Heavy Metal Contamination of Soils 

As a consequence of industrialization during the last centuries, the heavy metal 
concentration of soils has increased worldwide (Adriano 2001). Hot spots of 
soil contamination are located in areas of large industrial activities, where surrounding 
agricultural areas are affected by atmospheric deposition of heavy metals. Also, 
agricultural practices, such as the application of sewage sludge or phosphate fertilisers, 
has led to increased metal concentration in soils (Puschenreiter et al. 2005a). 

Metal contamination of soils may also derive from geogenic sources. These 
natural metalliferous soils are the classical habitats for metal- accumulating plants and 
may be divided into four different main groups, depending on the parent rock materi- 
als (see Section 15.2). Most of these naturally contaminated soils are quite infertile, 
which is particularly true for Ni-rich serpentine soils (which are characterized by low 
NPK levels and a low Ca:Mg ratio; Baker et al. 2000). Indigenous soil contamination 
may be restricted to either very small spots of only a few square meters or may affect 
large areas of several square kilometers such as those found in Cornwall and Devon 
(UK), where up to 700km 2 are contaminated with As (Mitchell and Barr 1995). 



15.2 Heavy Metal Tolerance and Hyperaccumulation 
in Plants 

Heavy metal tolerance mechanisms were summarized by Schat et al. (2000) and by 
Clemens (2001). Briefly, the simplest heavy metal tolerance strategy, termed 
"avoidance", consists in limited uptake into the plant body. However, a clear 
evidence for this mechanism was only found for some arsenite-tolerant plants. 
In most metal- tolerant higher plants, heavy metals are strongly retained in root 



Angela Sessitsch 

Austrian Research Centers GmbH, Dept. of Biore sources, A-2444 Seibersdorf, Austria 

e-mail: angela.sessitsch@arcs.ac.at 



P. Dion and C.S. Nautiyal (eds.), Microbiology of Extreme Soils. Soil Biology 13 3 17 

© Springer- Verlag Berlin Heidelberg 2008 



318 A. Sessitsch and M. Puschenreiter 

tissues. Regardless of whether heavy metals are mainly accumulated in roots or in 
shoots, internal tolerance mechanisms are the basis for efficient detoxification of 
the metals. This internal detoxification is based on (i) sequestration of the metals, 
that is, transport to cell components not involved in physiological processes 
(vacuole, cell wall); and (ii) complexation with metal-binding peptides, that is, 
metallothioneines and phytochelatins. 

Hyperaccumulator plants are able to take up large amounts of metals in their aerial 
tissues without showing any symptoms of toxicity. Plants accumulating >l,000mg 
kg -1 of Cu, Co, Cr, Ni, or Pb, or >1 0,000 mg kg -1 of Mn or Zn have been defined as 
hyperaccumulator species (Baker and Brooks 1989). An extended definition was 
provided by Baker and Whiting (2002), who claimed that the shoot/root or leaf/root 
ratio has to be >1, indicating a clear partitioning of metals to the shoots. 

More than 400 plant species have been identified as hyper accumulators, of 
which 75% are Ni hyperaccumulator s growing on ultramafic soils (Baker et al. 
2000). Hyperaccumulation of heavy metals is found throughout the whole plant 
kingdom in temperate as well as tropical climates, but is typically restricted to 
endemic species growing on mineralised soils and related rock types. The 
most important types of metalliferous soil hosting hyperaccumulator plants are (1) 
serpentine soils (enriched in Ni, Cr, Co), (2) "calamine" soils (enriched in Cd, Pb, 
Zn), (3) Se-rich soils, and (4) Cu- and Co-containing soils (Reeves and Baker 
2000). Related to the soil habitat, the hyperaccumulators are classified into 
accumulators of (1) Ni, (2) Cd, Pb, Zn, (3) Se, (4) Co, Cu, and (5) other elements 
(Al, As, Cr, Mn, Tl; Reeves and Baker 2000). Some examples for metal hyperaccu- 
mulating plants are shown in Table 15.1. 

Very effective translocation and storage processes have been described in metal 
hyperaccumulating plants (e.g., Lasat and Kochian 2000; Salt and Kramer 2000; 
Assungao et al. 2003). Metal sequestration into the vacuole or the apoplast and the 
complexation of metals by organic acids, amino acids, or specific high-affinity ligands 
are the basis for the high metal tolerance of hyperaccumulators (Salt and Kramer 
2000). To accumulate and be sequestered in plants, heavy metals must be taken up, 



Table 15.1 The highest observed foliar concentrations of As, Cd, Co, Cu, Ni, Pb, and Zn in 
metal-hyperaccumulating plants (Baker et al. 2000) 







Highest Observed Foliar 


Element 


Species 


Concentration (mg kg -1 dry weight) 


As 


Pteris vittata* 


23,000 


Cd 


Thlaspi caerulescens 


1,000 


Co 


Haumaniastrum robertii 


10,200 


Cu 


Aeollanthus biformifolius 


13,700 


Ni 


Sebertia acuminata 


260,000** 


Pb 


Thlaspi rotundifolium 


8,200 


Zn 


Thlaspi caerulescens 


35,000 



* Ma et al. (2001). 

** Found in the blue-green latex. 



15 Endophytes and Rhizosphere Bacteria of Plants Growing 



319 



for which high expression of metal transporter genes and active root proliferation 
towards contaminated soil spots have been reported as the main processes (Assungao 
et al. 2003). Additionally, some evidence for heavy metal mobilisation processes in 
the rhizosphere was reported (Puschenreiter et al. 2003, 2005b). 



15.3 Rhizosphere Bacteria and Endophytes 

Roots supply inorganic nutrients and water to the rest of the plant, whereas shoots 
fix carbon through photosynthesis and transport organic carbon compounds to the 
roots. The roots excrete a significant proportion of the transported carbon into the 
surrounding soil environment, which is biologically and biochemically influenced 
by the living root, known as the rhizosphere. The rhizosphere is a dynamic environ- 
ment and hosts a wide variety of micro-organisms. A schematic presentation of the 
distribution of microbes at the rhizoplane is shown in Fig. 15.1. Exudates released 
by plant roots and associated microbes may significantly mobilize heavy metals 
and thus increase their bioavailability (Wenzel et al. 1999). In addition, the apoplast 
of plants is commonly colonised by a wide range of bacterial endophytes that do 



Lateral 
roots 




• Root/ Microbial 
exudates 

Metals 

Compfexod 
metals C 

Clay minora! particfa 



Claw minora! oari 



Fig. 15.1 Distribution patterns of microbial populations and root exudates in the rhizosphere, (A) 
along the rhizoplane and (B) perpendicular to the rhizoplane; (C) mobilisation of mineral nutrients 
and heavy metals in the rhizosphere from the soil solid phase (e.g., clay minerals) by complexation 
with root and/or microbial exudates. After mobilisation (1), the complexed nutrients/metals are 
transported (2) to the root surface by mass flow and diffusion. (Compiled from Romheld 1991; 
Marschner 1995; Wenzel et al. 1999) 



320 A. Sessitsch and M. Puschenreiter 

not exhibit pathogenicity (Hallmann et al. 1997; Sturz and Nowak 2000; Reiter et 
al. 2002; Sessitsch et al. 2002; Idris et al. 2004). 

Some plant-associated micro-organisms are detrimental to plant health because they 
compete with the plant for nutrients or cause disease. However, a wide range of bacteria 
have a beneficial effect on plants. Some of these support plant resistance against patho- 
gens by either producing antibiotic substances or by inducing plant defenses. Other 
bacteria are able to stimulate plant growth by increasing the supply of nutrients to the 
plants or by producing plant growth hormones (Lugtenberg et al. 1991). 

Plant growth-promoting bacteria (PGPB) can directly promote plant growth by 
production of bacterial metabolites that positively affect the plant (Mahaffee and 
Kloepper 1994). Although the mechanisms still remain unclear, some reports indi- 
cate the production of phytohormones (de Freitas et al. 1990; Frommel et al. 1991) 
as being responsible for plant growth promotion. Many plant-associated bacteria 
synthesize the plant hormone and growth regulator indole-3-acetic acid (IAA) 
(Costacurta et al. 1994; Patten and Glick 1996), and enhanced production of IAA 
by a rhizosphere bacterium can improve plant growth (Beyeler et al. 1999). 
In addition, the production of cytokinin by rhizobacteria has also been suggested to 
enhance plant growth (Timmusk et al. 1999). The enzyme 1-aminocyclopropane- 
1-carboxylic acid (ACC) deaminase has been isolated from some bacteria (Campbell 
and Thompson 1996; Shah et al. 1998). This enzyme has no function in bacteria, but 
cleaves ACC, the precursor of ethylene in plants, and thus modulates ethylene levels, 
which can be particularly high under stress conditions. This enzymatic reaction 
contributes to plant growth promotion (Glick et al. 1997; Burd et al. 1998). 

Many PGPB suppress phytopathogens by mechanisms such as antibiosis or by 
inducing systemic resistance. A variety of biocontrol strains simply outcompete 
phytopathogens by efficiently colonizing plants (Dekkers et al. 2000; Chin- 
A-Woeng et al. 2000). In addition, the production of siderophores plays an impor- 
tant role in iron competition and has been identified as a mechanism contributing 
to biocontrol activity (Penyalver et al. 2001). Furthermore, it has been suggested 
that siderophores might be involved in the induction of systemic resistance (ISR) 
(De Meyer and Hofte 1997; Maurhofer et al. 1998). 

Bacterial endophytes have been defined as "bacteria, which for all or part of their 
life cycle invade the tissues of living plants and cause unapparent and asymptomatic 
infections entirely within plant tissues, but cause no symptoms of disease" (Wilson 
1995). Endophytes colonize a similar ecological niche as plant pathogens and may 
gain entry into plants by a number of mechanisms. Host entry points include tissue 
wounds (Agarwhal and Schende 1987; Lamb et al. 1996), stomata (Roos and Hattingh 
1983), lenticels (Scott et al. 1996), and germinating radicles (Gagne et al. 1987). 
Bacteria also may invade intact plants by penetrating root hair cells (Huang 1986) or 
by producing cell wall-degrading enzymes (Huang 1986; Quadt-Hallmann et al. 
1997). Endophytes including Proteobacteria, Firmicutes, and the Bacteroidetes 
phylum (Hallmann et al. 1997) have been studied mainly by cultivation-based methods 
(Bell et al. 1995; Stoltzfus et al. 1998; Sturz et al. 1998; Sessitsch et al. 2004). Results 
of recent endophyte analyses by cultivation-independent, 16S rRNA-based approaches 
indicated that individual plants host a broad phylogenetic range of endophytic bacteria 



15 Endophytes and Rhizosphere Bacteria of Plants Growing 321 

(Chelius and Triplet!, 2001; Sessitsch et al. 2002; Idris et al. 2004). By 16S rRNA gene 
analysis, it has been also demonstrated that biotic and abiotic stress may affect potato 
endophyte communities (Reiter et al. 2002; Sessitsch et al. 2002). Several studies 
suggested that the plant growth-promoting and biocontrol potential of endophytic 
bacteria is high as compared to that of rhizosphere microbes (van Buren et al. 1993; 
Reiter et al. 2002; Sessitsch et al. 2004). The mechanisms for biocontrol and growth 
promotion by an endophyte may be similar to those exhibited by rhizosphere bacteria. 



15.4 Diversity of Bacterial Communities Associated 
with Heavy Metal-Tolerant Plants 

Heavy metals affect the growth and activity of micro-organisms in soils mainly 
through destruction of the integrity of cell membranes, protein denaturation, and 
functional disturbance (Leita et al. 1995). Studies of the impact of heavy metals upon 
bacterial diversity in soils have shown mostly a negative influence (Hirsch et al. 1993; 
Smit et al. 1997; Sandaa et al. 2001; Moffett et al. 2003; Hinojosa et al. 2005). Other 
factors such as pH, temperature, moisture, and organic matter content may interfere 
with metal toxicity (Giller et al. 1998). It is well known that bacteria and fungi isolated 
from polluted habitats are tolerant of higher levels of metals than those isolated from 
unpolluted areas (Baath et al. 1989; Doelman et al. 1994; Huysman et al. 1994; 
Mertens et al. 2006). After the addition of metals, metal tolerance is increased in 
bacterial communities by the death of sensitive species and subsequent competition 
and adaptations of surviving bacteria (Diaz-Rovina et al. 1996). Horizontal transfer of 
plasmids containing resistance genes may greatly contribute to the adaptation process. 
It has been shown that microbial biomass and enzyme activities are more sensitive to 
heavy metal contamination than is species diversity (Kandeler et al. 2000). 

Concentrations and bioavailabilities of heavy metals, and therefore their toxicity 
to the rhizosphere microflora, might be altered in the rhizosphere. Roots may 
absorb heavy metals to a certain extent. In addition, root exudates may either 
complex metals, making them unavailable to microbes, or enhance metal release, 
for example, by altering the pH. Contrasting results regarding the ability of root 
excretions to mobilize heavy metals have been reported, showing either an increased 
mobility (Morel et al. 1986; Bernal and McGrath 1994; Cieslinski et al. 1998; 
Fitz et al. 2003; Wenzel et al. 2003; Puschenreiter et al. 2005b) or no effect 
(Whiting et al. 2001a; Zhao et al. 2001; Amir and Pineau, 2003). 

Due to their importance for practical applications, plant-microbe interactions 
involving plant species with some relevance to phytoremediation have been the 
object of particular attention. Kunito et al. (2001) compared the characteristics of 
bacterial communities in the rhizosphere of Phragmites with those of nonrhizo- 
sphere soil in a highly Cu-contaminated area near a copper mine in Japan. Phragmites 
is an important plant for phytoremediation applications, but does not hyperaccumu- 
late heavy metals. Higher bacterial numbers were detected in the rhizosphere, which 
may be due to the lower Cu concentrations or to the availability of root exudates. 



322 A. Sessitsch and M. Puschenreiter 

Nevertheless, the percentage of highly resistant strains was higher in the rhizosphere 
than in nonrhizosphere soil. Cu toxicity was found to be lower in rhizosphere soil. 
The study by Kunito et al. (2001) also indicated that Cu toxicity lowered the 
frequency of r-strategists (bacteria capable of rapid growth and utilization of 
resources) as these are more sensitive to toxic substances (Kozdroj 1995). 

Rhizosphere and nonrhizosphere isolates behaved very differently regarding their 
doubling time and exopolymer production (Kunito et al. 2001). Exopolymers 
produced by bacteria were shown to strongly bind heavy metals (Bitton and Freihofer 
1978), leading to the formation of organic-metal complexes, which are difficult to 
degrade (Francis et al. 1992; Hattori et al. 1996; Huysman et al. 1994). Furthermore, 
heavy metal concentrations induce the production of exopolymer production (Chao 
and Chen 1991; Kidambi et al. 1995) and metal resistance due to exopolymer 
production has been shown (Bitton and Freihofer 1978). Kunito et al. (1997) reported 
a dominance of Cu-resistant Bacillus spp. in the rhizosphere of Phragmites, whereas 
nonrhizosphere soil was dominated by Methylobacterium spp. 

In the rhizosphere of heavy metal hyperaccumulating plants, higher proportions 
of resistant bacteria were found (Schlegel et al. 1991; Mengoni et al. 2001). 
Mengoni et al. (2001) sampled soils from three Ni-containing, serpentine sites in 
Italy and found an increasing number of resistant strains in increasing proximity 
to the Ni-hyperaccumulating plant, Alyssum bertolonii. At all sites, the culturable 
Ni-resistant rhizosphere community was dominated by Pseudomonas, whereas soil 
samples contained a high number of Ni-resistant Streptomyces spp. In general, iso- 
lates obtained in this study showed co-resistance to Cr and Co, although other 
resistance combinations (e.g., Ni, Zn, and Cu) or single resistance were also found, 
indicating independent evolution of heavy metal resistance determinants. Mengoni 
et al. (2004) analyzed the same sites by cultivation-independent analysis, for which 
soil samples at different distances to the Alyssum bertolonii roots were examined. 
Results showed that the plant rather than the locality shaped the microbial community 
structure. In agreement with the cultivation-dependent analysis, proteobacteria 
were predominantly found in the rhizosphere. 

As was the case for the Alyssum bertolonii rhizosphere (see above, this section), 
a high percentage of proteobacteria was found in the rhizosphere of the 
Ni-hyperaccumulator Thlaspi goesingense by cultivation-independent analysis. In 
addition, T. goesingense hosted members of the phylum Holophaga/Acidobacterium, 
high-GC Gram positive bacteria, members of the Bacteroidetes phylum and Verruco- 
microbia (Idris et al. 2004). Cultivation of bacteria on Ni-containing medium resulted 
mostly in the isolation of Methylobacterium spp., an alpha-proteobacterial genus, as 
well as Rhodococcus spp. and Okibacterium spp., belonging to the Actinobacteria (or 
Gram-positive bacteria with a high G+C content; Idris et al. 2004). 

Methylobacteria were found to be dominant as endophytic colonizers of 
Thlaspi goesingense (Idris et al. 2004). Although Methylobacterium extorquens 
and Methylobacterium mesophilicum were both identified in the rhizosphere as 
well as inside the plant, different strains were found suggesting that these habitats 
provide distinct growth conditions for micro-organisms. One strain isolated from 



15 Endophytes and Rhizosphere Bacteria of Plants Growing 323 

Thlaspi goesingense shoots fell into a newly described species, Methylobacterium 
goesingense (Idris et al. 2006). M. extorquens, M. mesophilicum, and M. goesingense 
strains were highly resistant to Ni, but were also shown to be resistant against 
different combinations of heavy metals, indicating the independent evolution of 
resistance traits. Despite the fact that heavy metal determinants are frequently 
located on plasmids, horizontal transfer of plasmids between Methylobacterium 
spp. isolated from Thlaspi goesingense shoots and rhizosphere was found to be 
unlikely (Idris et al. 2006). 

A large number of heavy metal tolerating methylobacteria were also isolated 
from shoots of the Zn-hyperaccumulator Thlaspi caerulescens, but they were not 
found in association with roots (Lodewyckx et al. 2002). Striking was the high 
diversity of endophytes colonizing Thlaspi goesingense, as revealed by cultiva- 
tion-independent analysis (Idris et al. 2004). Bacteria belonging to all major 
bacterial phyla could be found, including Acidobacteria. The phylum Holophaga/ 
Acidobacterium has been found to be dominant in many soils worldwide (e.g., 
Sessitsch et al. 2001) and Acidobacteria may also colonize the rhizosphere 
(e.g., Idris et al. 2004; Mengoni et al. 2004). However, usually these bacteria do 
not colonize the apoplast of plants. 

In the highly toxic environment represented by the apoplast of a Ni- 
hyperaccumulating plant, the diversity of endophytes was higher than that usually 
observed among endophytes from other plants (e.g., Rasche et al. 2006; Reiter and 
Sessitsch 2006). Lodewyckx et al. (2002) compared root and rhizoplane isolates 
with endophytes isolated from Thlaspi caerulescens shoots. Similar species were 
found in both compartments. However, shoot endophytes showed higher resistance 
to Zn and Cd than strains isolated from roots and rhizoplane. Most isolates from the 
Zn hyperaccumulators were affiliated with the genera Methylobacterium and 
Sphingomonas, which were also found to be represented in high numbers in shoots 
of the Ni hyperaccumulator Thlaspi goesingense (Idris et al. 2004) as well as in 
Zn accumulating willows (Kuffner et al. unpublished results). 

Cultivation-independent 16S rDNA-based analysis of bacteria has revolu- 
tionized our understanding of bacterial diversity, as micro-organisms can be 
investigated irrespectively of their culturability. However, DNA-based analysis 
does not give any information on the activity of cells, and even dead cells may 
be detected insofar as their DNA has not been destroyed by nuclease activity. 
As metabolically active cells usually contain a higher amount of ribosomes than 
resting or dormant cells, Gremion et al. (2003) used 16S rDNA as well as 16S 
rRNA-based analysis to characterize bacterial diversity in heavy metal- 
contaminated bulk soil and in the rhizosphere of Thlaspi caerulescens. DNA- 
based analysis indicated the dominance of proteobacteria, acidobacteria, and 
planctomycetes in the rhizosphere. A minority of bacteria were affiliated with 
the high-GC Gram-positive bacteria (actinobacteria) and verrucomicrobia.In 
contrast, analysis of the metabolically active population showed that members 
of the Rubrobacteria subdivision, belonging to the high-GC Gram-positive 
bacteria, were highly dominating. 



324 A. Sessitsch and M. Puschenreiter 

15.5 The Influence of Associated Micro-Organisms 
on Plant Heavy Metal Tolerance and Uptake 

The analysis of 40 ultramafic soil samples from New Caledonia identified a high 
positive correlation between bioavailability of heavy metals and microbial activity, 
suggesting a possible role of micro-organisms in the release of heavy metals (Amir 
and Pineau 2003). These findings were confirmed by an experiment in which 
autoclaved soil was inoculated with a small portion of the same nonheated soil, leading 
to the release of metals (Amir and Pineau 2003). The effect of pH was found to be 
marginal, whereas the presence of a heavy metal-tolerant plant as well as the addition 
of compost stimulated the metal mobilization process. Amir and Pineau (2003) 
suggested that the rhizosphere effect on metal release is due to the stimulation of che- 
moorganotrophic micro-organisms rather than to the direct effect of root secretions. 

The effect of root exudates on metal mobilization was reported by Bernal and 
McGrath (1994). Amir and Pineau (2003) did not exclude the possibility that roots 
would absorb a part of the released metals. Several studies have shown that 
rhizosphere bacteria may contribute to the plant metal tolerance and increase metal 
uptake. Stimulation of plant growth and root biomass was most probably responsible 
for the increased heavy metal uptake by Brassica juncea in the presence of heavy 
metal-tolerant bacteria (Salt et al. 1999). de Souza et al. (1999a) showed that rhizo- 
sphere bacteria are necessary to achieve optimum rates of selenium accumulation 
and volatilization by Indian mustard. In this study, the tested rhizosphere bacteria 
increased root hair production of Indian mustard and enhanced metal uptake was 
observed. However, from the different experiments performed the authors concluded 
that stimulation of root hair production was not responsible for the enhanced 
accumulation of Se. A heat-labile compound was shown to enhance Se accumula- 
tion in axenic plants, but it was not clear whether it was produced by rhizosphere 
bacteria or by bacterized roots (de Souza et al. 1999a). The authors postulated that 
the heat-labile compound stimulated the selenate transporter in plants. 

Whiting et al. (2001b) studied the effect of rhizosphere bacteria on Zn accumulation 
by the hyperaccumulating plant Thlaspi caerulescens and the nonaccumulator 
T. arvense. They showed that bacteria facilitated biomass production and Zn uptake 
of the hyperaccumulator, whereas T. arvense was not affected. Furthermore, significantly 
higher microbial numbers were detected in the rhizosphere of T. caerulescens than in 
that of T. arvense, which was probably a result of the higher root biomass of the 
accumulating species. Nevertheless, it was shown that the increased uptake of Zn was 
not a result of increased root surface area. Data suggested that the bacteria enhanced 
the availability of water-soluble Zn in the soil, which overcame a major rate-limiting 
step for Zn uptake by T. caerulescens in soils with low concentrations of labile Zn 
(Whiting et al. 2001b). Similarly, rhizobacteria were considered as highly important 
for the mobilization of nickel in soil and for its uptake by the hyperaccumulating plant 
Alyssum murale (Abou-Shanab et al. 2003). Our own studies revealed a strong effect 
on accumulated heavy metal contents in willows due to plant growth-promoting 
bacteria (Kuffner et al. unpublished results). 



15 Endophytes and Rhizosphere Bacteria of Plants Growing 325 

Apart from plant growth promotion, which may be achieved by a range of bacte- 
rial activities such as the production of hormones or the provision of nutrients, 
micro-organisms may improve the stress tolerance of the plant. This may be 
achieved by the enzyme ACC deaminase leading to a reduction of stress-induced 
ethylene levels in the plant (Burd et al. 1998, 2000; Glick 2004). Furthermore, 
bacteria may stimulate the production of metal transporters in plants (de Souza 
et al. 1999a,b). Heavy metal mobilization was proposed to be due to the action of 
bacterial siderophores (Lodewyckx et al. 2002; Abou-Shanab et al. 2003). These 
compounds show high affinity for ferric iron but also form complexes with bivalent 
heavy metal ions (Evers et al. 1989) that can be assimilated by the plant. 
Furthermore, heavy metals have been shown to stimulate the production of bacte- 
rial siderophores (van der Lelie et al. 1999). 

In addition to these direct effects, bacterial siderophores can indirectly alleviate 
heavy metal toxicity by increasing the supply of iron to the plant (Burd et al. 1998, 
2000). Our own studies showed that siderophore production is a frequently found 
trait among bacteria associated with heavy metal accumulating plants (Idris et al. 
2004; Kuffner et al. unpublished results). However, those siderophore-producing 
bacteria which were tested for their potential to support heavy metal uptake did not 
show the expected effects. On the other hand, several strains that did not produce 
siderophores mobilized high amounts of heavy metals, probably due the production 
of other secondary metabolites (Kuffner et al. unpublished results). In addition to 
the potential effect of siderophores it has been suggested that bacterial exopolymers 
may complex heavy metals leading to reduced availablity for plants (Diels et al. 
1995; Kunito et al. 2001). 



15.6 Potential Applications of Improved 
Plant-Microbe Interactions 

Various studies demonstrating that plant-associated micro-organisms greatly con- 
tribute to the mobilization and accumulation of heavy metals as well as to the stress 
resistance of plants suggest that appropriate strains may be inoculated in order to 
further improve phytoextraction applications. Initial studies have been performed 
by Whiting et al. (2001b), who inoculated Thlaspi caerulescens seeds grown in 
sterile as well as in nonsterile soil with rhizosphere bacteria belonging to the 
species Microbacterium saperdae, Pseudomonas monteilii, and Enterobacter 
cancerogenes. Bacteria enhanced Zn uptake in sterile soils. However, in nonsterile 
soils the effect of the bacterial inoculant strains was masked by the natural soil 
microflora. This indicates that several native strains have the capability to enhance 
heavy metal mobilization. When inoculated, superior strains, which are promising 
as inoculants for enhancing phytoextraction, usually encounter high numbers of 
highly adapted micro-organisms and therefore have to show high competitive abilities. 
This is particularly true for rhizosphere bacteria, whereas endophytes usually 
encounter less competition. 



326 A. Sessitsch and M. Puschenreiter 

Endophytic bacteria with good colonization potential were endowed with 
heavy metal resistance using genetic methods. The ncc-nre nickel resistance 
system of Cupriavidus metallidurans (previously Ralstonia metallidurans) was 
efficiently expressed in two endophyte strains of the species Burkholderia cepacia 
and Herbaspirillum seropedicae (Lodewyckx et al. 2001). When inoculated 
onto Lupinus luteus L. plants, the Ni-resistant B. cepacia strain induced a 
significant increase of Ni concentrations in roots but not in shoots. Similarly, 
the Ni-resistant Herbaspirillum strain enhanced Ni uptake of Lolium perenne 
plants; however, the wild-type bacterial strain showed the same effect, indicating 
that nickel resistance was not responsible for the effect observed with the 
recombinant Herbaspirillum strain (Lodewyckx et al. 2001). Recombinant strains 
of the rhizosphere bacterium Pseudomonas aureofaciens were engineered by 
either adding the arsenite resistance operon or the citrate synthase gene of 
Pseudomonas aeruginosa PA01 (Sizova et al. 2004). The latter genetically 
modified strain mobilized arsenic in soils, whereas the former was highly resistant 
to arsenite and arsenate. Both strains increased the survival of sorghum 
{Sorghum saccharatum L.) plants and led to highly increased levels of plant- 
accumulated arsenic. 

Genetic engineering of micro-organisms and/or plants for improved phytoex- 
traction has been also proposed by Sauge-Merle et al. (2003). As phytochelatins 
(PCs) bind heavy metals by complex formation, the authors expressed PC synthase 
of Arabidop sis thaliana in Escherichia coli. Significant increases of cellular heavy 
metal content were found and the authors suggest using genes of the PC biosyn- 
thetic pathway for the design of bacterial strains or higher plants with increased 
abilities to accumulate toxic metals. 



15.7 Conclusions 

Rhizosphere and endophytic bacteria are associated with plants showing tolerance 
to heavy metals and may modulate this tolerance. Endophytic colonizers may pos- 
sess a better potential at influencing plant activities, as compared to rhizospheric 
micro-organisms. The structure of plant-associated bacterial communities is influ- 
enced by heavy-metal soil contamination, with resistant strains being selected. 
Rhizosphere and endophytic bacteria may increase the capacity of the plant to 
hyperaccumulate heavy metals by a variety of direct and indirect mechanisms. 
Direct mechanisms include enhanced heavy-metal mobilization and an allevation 
of heavy-metal toxicity to the plant by improving iron nutrition. Indirect mecha- 
nisms comprise plant growth promotion and improved stress tolerance. 

These effects of rhizosphere and endophytic micro-organisms suggest that specific 
strains with good activity and colonization potential would be useful in enhancing 
phytoextraction applications. In particular, the application of genetically engineered 
plant-associated micro-organisms may be a promising approach for phytoremediation 
of soils contaminated with heavy metals. However, the performance of these 



15 Endophytes and Rhizosphere Bacteria of Plants Growing 327 

micro-organisms under natural conditions has to be investigated in detail. Although 
these strains are likely to be superior in terms of heavy metal resistance and mobi- 
lization, they might face competition problems similar to promising natural strains. 
Furthermore, biosafety aspects have to be considered and their release depends on 
national legislation. Addressing the issue of persistence and competition capacity 
of inoculant strains, while developing their potential for plant growth promotion, 
stress resistance, and heavy metal accumulation, represent promising strategies for 
improving current phytoremediation techniques. 



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